Novel proteins specific for pyoverdine and pyochelin

ABSTRACT

The present disclosure provides hNGAL muteins that bind a pyoverdine family member or pyochelin and can be used in various application including pharmaceutical applications, for example, to inhibit or reduce growth of P. aeruginosa. The present disclosure also concerns methods of making one or more pyoverdine- or pyochelin-binding muteins described herein as well as compositions comprising one or more of such muteins. The present disclosure further relates to nucleic acid molecules encoding such muteins and to methods for generation of such muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these muteins as well as compositions comprising one or more of such muteins.

I. BACKGROUND

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen thatcauses acute infections, primarily in association with tissue injuries.P. aeruginosa forms biofilms on indwelling devices and on the pulmonarytissues of patients with the genetic disorder, cystic fibrosis. Biofilminfections are difficult to treat with conventional antibiotictherapies. However, research has demonstrated that iron is essential forproper biofilm formation by P. aeruginosa, and therefore iron-uptakesystems are potential targets for anti-Pseudomonas therapies.

P. aeruginosa is able to scavenge iron from the host environment byusing the secreted iron-binding siderophores, pyochelin and pyoverdine.Pyoverdine (Pvd) is a peptide-linked hydroxamate- and catecholate-typeligand, and pyochelin (Pch) a derivatized conjugate of salicylate andtwo molecules of cysteine and having phenol, carboxylate, and amineligand functionalities. Both Pvd and Pch have demonstrated roles in P.aeruginosa virulence with some indication of synergism. Double-deficientmutants unable to make either siderophore are much more attenuated invirulence than either single-deficient mutant unable to make just one ofthe two siderophores (Takase et al., Infection and immunity, April 2000,p. 1834-1839). Furthermore, pyoverdine acts as a signalling molecule tocontrol production of several virulence factors as well as pyoverdineitself; while it has been proposed that pyochelin may be part of asystem for obtaining divalent metals such as ferrous Iron and zinc forP. aeruginosa's pathogenicity, in addition to ferric iron (Visca et al.,1992).

Three structurally different pyoverdine types or groups have beenidentified from several P. aeruginosa strains: from P. aeruginosa ATCC15692 (Briskot et al., 1989, Liebigs Ann Chem, p. 375-384), from P.aeruginosa ATCC 27853 (Tappe et al., 1993, J. Prakt-Chem., 335, p.83-87) and from a natural isolate, P. aeruginosa R (Gipp et al., 1991,Z. Naturforsch, 46c, p. 534-541). Moreover, comparative biologicalinvestigations on 88 clinical isolates and the two collection strainsmentioned above revealed three different strain-specificpyoverdine-mediated iron uptake systems (Comells et al., 1989, InfectImmun., 57, p. 3491-3497; Meyer et al., 1997, Microbiology, 143, p.35-43) according to the reference strains: P. aeruginosa ATCC 15692(Type I Pvd or Pvd I), P. aeruginosa ATCC 27853 (Type II Pvd or Pvd II)and the clinical isolates P. aeruginosa R and pa6 (Type III Pvd or PvdIII).

Each pyoverdine type has three members (subtypes) differing in the sidechain which is succinyl, succinamid or α-ketoglutaryl, namely, Pvd typeI succinyl, Pvd type I succinamid, Pvd type I α-ketoglutaryl, Pvd typeII succinyl, Pvd type II succinamid, Pvd type II α-ketoglutaryl, Pvdtype III succinyl, Pvd type III succinamid and Pvd type IIIα-ketoglutaryl.

Each P. aeruginosa strain expresses one Pvd type i.e. P. aeruginosa ATCC15692 expresses Type I Pvd, P. aeruginosa ATCC 27853 expressesType IIPvd and P. aeruginosa R and pa6 expressesType III Pvd, whereby each Pvdtype includes all three members of the respective type, and each saidstrain also expresses pyochelin.

In this regard, we identified the pyoverdins and pyochelin as targetswhich are crucial for P. aeruginosa's pathogenicity and developedspecific inhibitors for such targets, as disclosed here, i.e. for eachtype of Pvd including for every type the three members (subtypes)differing in the side chain (Pvd I s, Pvd I sa, Pvd I αKG, Pvd II s, PvdII sa, Pvd II αKG, Pvd III s, Pvd III sa, Pvd III αKG) as well as forPch, and in every case to the free siderophore as well as to thesiderophore with bound iron without creating the strong selectivepressure imposed by conventional antibiotics. In addition, we selectedinhibitors that distinguish free and iron-loaded pyochelin.

The present invention was made as a result of activities undertaken onbehalf of Pieris AG, Sanofi-Aventis and Sanofi-Pasteur Inc., which areparties to an existing joint research agreement, and was made within thescope of the joint research agreement.

II. DEFINITIONS

The following list defines terms, phrases, and abbreviations usedthroughout the instant specification. All terms listed and definedherein are intended to encompass all grammatical forms.

As used herein, “pyoverdine” means a fluorescent siderophore that isproduced by the gram negative bacterium Pseudomonas aeruginosa underiron-deficient growth conditions and has high affinity for iron.Pyoverdines are composed of three structural parts: a dihydroxyquinolinechromophore, a side chain and a variable peptidic chain.

The peptide chain moiety is involved in receptor recognition andbinding. Three different Pvds, differing in their peptide chain, havebeen identified (types I-III). The size and amino acid composition ofpyoverdine types are unique to each species, as well as the pyoverdinerecognition specificity. Three P. aeruginosa strains can bedistinguished, each producing a different pyoverdine type (type I-III,FIG. 1) and a cognate FpvA receptor.

As used herein, “pyochelin” means a thiazoline derivatized conjugate ofsalicylate and two molecules of cysteine and having phenol, carboxylate,and amine ligand functionalities, produced by P. aeruginosa andsolubilizing ferric iron. Pyochelin is a structurally unique siderophorepossessing phenolate, but neither a hydroxamate nor a catecholate moiety(see FIG. 1.)

As used herein, “detectable affinity” means the ability to bind to aselected target with an affinity constant of generally at least about10⁻⁵ M or below. Lower affinities are generally no longer measurablewith common methods such as ELISA and therefore of secondary importance.

As used herein, “binding affinity” of a protein of the disclosure (e.g.a mutein of human lipocalin 2) or a fusion polypeptide thereof to aselected target (in the present case, pyoverdine or pyochelin), can bemeasured (and thereby KD values of a mutein-ligand complex bedetermined) by a multitude of methods known to those skilled in the art.

Such methods include, but are not limited to, fluorescence titration,direct ELISA, competition ELISA, calorimetric methods, such asisothermal titration calorimetry (ITC), and surface plasmon resonance(BIAcore). Such methods are well established in the art and examplesthereof are also detailed below.

It is also noted that the complex formation between the respectivebinder and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(D) (for example fluorescence titration, direct ELISA,competition ELISA or surface plasmon resonance, just to name a few) oreven the mathematical algorithm which is used for evaluation of theexperimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivebinder and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular mutein for a given ligand. Thismeans that there may be a slight deviation in the measured K_(D) valuesor a tolerance range depending, for example, on whether the K_(D) valuewas determined by surface plasmon resonance (Biacore), by competitionELISA, or by “direct ELISA.”

As used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid), or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, compared to thenaturally occurring (wild-type) nucleic acid or protein “reference”scaffold. Said term also includes fragments of a mutein and variants asdescribed herein. Muteins of the present disclosure, fragments orvariants thereof preferably retain the function of binding to pyoverdineor pyochelin as described herein.

The term “fragment” as used herein in connection with the muteins of thedisclosure relates to proteins or peptides derived from full-lengthmature human lipocalin 2 that are N-terminally and/or C-terminallyshortened, i.e. lacking at least one of the N-terminal and/or C-terminalamino acids. Such fragments may include at least 10, more such as 20 or30 or more consecutive amino acids of the primary sequence of the maturehuman lipocalin 2 and are usually detectable in an immunoassay of themature human lipocalin 2. In general, the term “fragment”, as usedherein with respect to the corresponding protein ligand of a mutein ofthe disclosure or of the combination according to the disclosure or of afusion protein described herein, relates to N-terminally and/orC-terminally shortened protein or peptide ligands, which retain thecapability of the full length ligand to be recognized and/or bound by amutein according to the disclosure.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the mature human lipocalin 2 can besubstituted by at least one amino acid that is not present at thisspecific position in the respective natural polypeptide sequence. Theterm “mutagenesis” also includes the (additional) modification of thelength of sequence segments by deletion or insertion of one or moreamino acids. Thus, it is within the scope of the disclosure that, forexample, one amino acid at a chosen sequence position is replaced by astretch of three random mutations, leading to an insertion of two aminoacid residues compared to the length of the respective segment of thewild type protein. Such an insertion or deletion may be introducedindependently from each other in any of the peptide segments that can besubjected to mutagenesis in the disclosure.

The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated with a certain probability ata predefined sequence position during mutagenesis.

“Identity” is a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent disclosure means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the disclosure with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Sequence identity ismeasured by dividing the number of identical amino acid residues by thetotal number of residues and multiplying the product by 100.

The term “homology” is used herein in its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby an aspartate residue) at equivalent positions in the linear aminoacid sequence of a polypeptide of the disclosure (e.g., any mutein ofthe disclosure).

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1; cutoff value set to 10⁻³) including the propeptidesequences, preferably using the wild type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a mutein different from the wild-type humanlipocalin 2 corresponds to a certain position in the amino acid sequenceof the wild-type human lipocalin 2, a skilled artisan can use means andmethods well-known in the art, e.g., alignments, either manually or byusing computer programs such as BLAST2.0, which stands for Basic LocalAlignment Search Tool or ClustalW or any other suitable program which issuitable to generate sequence alignments. Accordingly, the wild-typehuman lipocalin 2 can serve as “subject sequence” or “referencesequence”, while the amino acid sequence of a mutein different from thewild-type human lipocalin 2 described herein serves as “query sequence”.The terms “reference sequence” and “wild type sequence” are usedinterchangeably herein.

“Gaps” are spaces in an alignment that are the result of additions ordeletions of amino acids. Thus, two copies of exactly the same sequencehave 100% identity, but sequences that are less highly conserved, andhave deletions, additions, or replacements, may have a lower degree ofsequence identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity usingstandard parameters, for example Blast (Altschul, et al. (1997) NucleicAcids Res. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol.215, 403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.147, 195-197).

The term “variant” as used in the present disclosure relates toderivatives of a protein or peptide that include modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Such modifications do in some embodiments notreduce the functionality of the protein or peptide. Such variantsinclude proteins, wherein one or more amino acids have been replaced bytheir respective D-stereoisomers or by amino acids other than thenaturally occurring 20 amino acids, such as, for example, omithine,hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.However, such substitutions may also be conservative, i.e. an amino acidresidue is replaced with a chemically similar amino acid residue.Examples of conservative substitutions are the replacements among themembers of the following groups: 1) alanine, serine, and threonine; 2)aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan.

By a “native sequence” human lipocalin 2 is meant human lipocalin 2 thathas the same amino acid sequence as the corresponding polypeptidederived from nature. Thus, a native sequence human lipocalin 2 can havethe amino acid sequence of the respective naturally-occurring humanlipocalin 2. Such native sequence polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the human lipocalin2, naturally-occurring variant forms such as alternatively spliced formsand naturally-occurring allelic variants of human lipocalin 2. Apolypeptide “variant” means a biologically active polypeptide having atleast about 50%, 60%, 70%, 80% or at least about 85% amino acid sequenceidentity with the native sequence polypeptide. Such variants include,for instance, polypeptides in which one or more amino acid residues areadded or deleted at the N- or C-terminus of the polypeptide. Generally avariant has at least about 70%, including at least about 80%, such as atleast about 85% amino acid sequence identity, including at least about90% amino acid sequence identity or at least about 95% amino acidsequence identity with the native sequence polypeptide.

The term “position” when used in accordance with the disclosure meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein. To understand the term “correspond” or“corresponding” as used herein in the context of the amino acid sequencepositions of one or more muteins, a corresponding position is not onlydetermined by the number of the preceding nucleotides/amino acids.Accordingly, the position of a given amino acid in accordance with thedisclosure which may be substituted may vary due to deletion or additionof amino acids elsewhere in a (mutant or wild-type) human lipocalin 2.Similarly, the position of a given nucleotide in accordance with thepresent disclosure which may be substituted may vary due to deletions oradditional nucleotides elsewhere in a mutein or wild type humanlipocalin 2 5′-untranslated region (UTR) including the promoter and/orany other regulatory sequences or gene (including exons and introns).

Thus, for a corresponding position in accordance with the disclosure, itis preferably to be understood that the positions of nucleotides/aminoacids may differ in the indicated number than similar neighbouringnucleotides/amino acids, but said neighbouring nucleotides/amino acids,which may be exchanged, deleted, or added, are also comprised by the oneor more corresponding positions.

In addition, for a corresponding position in a mutein based on areference scaffold in accordance with the disclosure, it is preferablyto be understood that the positions of nucleotides/amino acids arestructurally corresponding to the positions elsewhere in a mutein orwild-type human lipocalin 2, even if they may differ in the indicatednumber.

The term “organic molecule” or “small organic molecule” as used hereinfor the non-natural target denotes an organic molecule comprising atleast two carbon atoms, but preferably not more than 7 or 12 rotatablecarbon bonds, having a molecular weight in the range between 100 and2000 Dalton, preferably between 100 and 1000 Dalton, and optionallyincluding one or two metal atoms.

The word “detect”, “detection”, “detectable” or “detecting” as usedherein is understood both on a quantitative and a qualitative level, aswell as a combination thereof. It thus includes quantitative,semi-quantitative and qualitative measurements of a molecule ofinterest.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, rats, pigs, apes such as cynomolgous monkeys andetc., to name only a few illustrative examples. Preferably, the mammalherein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

A “sample” is defined as a biological sample taken from any subject.Biological samples include, but are not limited to, blood, serum, urine,feces, semen, or tissue.

III. DESCRIPTIONS OF FIGURES

FIG. 1A-FIG. 1E: shows the structure of P. aeruginosa siderophores. FIG.1A-C show the structures of the three P. aeruginosa pyoverdines. FIG.1A: Structure of Pvd type I Birskot et al., 1989); FIG. 1B: Structure ofPvd type II (see Birskot et al., 1989): FIG. 1C: Structure of Pvd typeIII (Gipp et al., 1991); FIG. 1 D: R attached to the chormophore partcan be a succinyl, succinamid or α-ketoglutaryl side chain; and FIG. 1E: Structure of pyochelin (Brandel et al., 2011).

FIG. 2A-FIG. 2H: provides typical measurements of on-rate and off-rateby Surface Plasmon Resonance for Pvd I s (+Fe) binding to the lipocalinmutein SEQ ID NO: 16 (FIG. 2A), Pvd II s (+Fe) binding to the lipocalinmutein SEQ ID NO: 36 (FIG. 2B), Pvd III (+Fe) binding to the lipocalinmutein SEQ ID NO: 53 (FIG. 2C) and Pyochelin (+Fe) binding to SEQ ID NO:62 (FIG. 2D). In addition, absence of binding of the respectivesiderophores at 1200 nM (200 nM for Pyochelin) to the negative controllipocalin SEQ ID NO: 64 is shown in FIG. 2E-FIG. 2H.

FIG. 3: shows an exemplary specificity and crossreactivity profile forthe lipocalin mutein SEQ ID NO: 35 as determined by Surface PlasmonResonance. Specific binding to Pyoverdin II succinyl, succinamid andα-ketoglutaryl is demonstrated, while absence of binding to Pyoverdinesof type I and type III. Pyochelin, Enterobactin and Desferoxamin isshown. High concentrations of 2 μM are used for all analytes.

FIG. 4A-FIG. 4D: shows exemplary data from growth inhibition assay. FIG.4A: Pvd I specific mutein SEQ ID NO: 16 shows growth inhibition of a PvdI specific P. aeruginosa strain (ATCC27853) compared to the controlculture growing without mutein. FIG. 4B: Pvd II specific muteins SEQ IDNOs: 19 and 36 show growth inhibition of a Pvd II specific P. aeruginosastrain (ATCC 15692) compared to the control culture growing withoutmutein. SEQ ID NO: 36 has a higher binding affinity compared to SEQ IDNO: 19 and shows a greater growth inhibition. FIG. 4C: Pvd III specificmutein SEQ ID NO: 53 shows growth inhibition of a Pvd III specific P.aeruginosa strain (ATCC33360) compared to the control culture growingwithout mutein. FIG. 4D: Pch specific muteins SEQ ID NO: 62 shows growthinhibition of a Pvd I knock out P. aeruginosa strain (ATCC15692 ΔpvdA)relying on Pch for iron uptake compared to the control culture growingwithout mutein. 10 μM lipocalin muteins were applied in the assay.

FIG. 5: shows in a P. aeruginosa-induced lung infection model in micethat administration of SEQ ID NO: 19, 1 hour before and at time ofbacteria challenge, prevents the development of infection in mice in adose-dependent manner. A significant prevention effect was observedstarting from SEQ ID NO: 19 at 200 μg/mouse, with a maximal effect at2000 μg/mouse.

FIG. 6: shows the amino acid sequence expressed for crystallisationincluding a start methionine at position 1, a lysine at postion 2, ahexahistidine tag at postion 3-8, a linker region of amino acids DYDIPTTat postion 9-15 (SEQ ID NO: 132), the tobacco etch viral (TEV) proteasecleavage site ENLYFQG at position 16-22 (SEQ ID NO: 133) followed by theamino acid sequence of the mutein of interest from position 23 onwards.

FIG. 7: shows the SEQ ID NO: 31—Pvd-Fe complex structure. An overlay oftwo SEQ ID NO: 31 molecules i.e. chain A and chain B from an asymmetricunit.

FIG. 8: shows SEQ ID NO: 31 and Pvd-Fe interactions. Two molecules fromasymmetric unit are overlaid. Side chains interacting with Pvd-Fe aredepicted.

FIG. 9: shows the Pvd composition. Oxygen atoms involved in iron bindingare boxed.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

The current disclosure provides a polypeptide having binding specificityfor pyoverdine type I, II, III or pyochelin, wherein the polypeptidecomprises an hNGAL mutein that binds pyoverdine type I, II, III orpyochelin with detectable affinity.

The term “human lipocalin 2” or “human Lcn 2” or “human NGAL” or “hNGAL”as used herein refers to the mature human neutrophilgelatinase-associated lipocalin (NGAL) with the SWISS-PROT/UniProt DataBank Accession Number P80188. A human lipocalin 2 mutein of thedisclosure may also be designated herein as “an hNGAL mutein”. The aminoacid sequence shown in SWISS-PROT/UniProt Data Bank Accession NumberP80188 may be used as a preferred “reference sequence”, more preferablythe amino acid sequence shown in SEQ ID NO: 1 is used as referencesequence.

In some embodiments, an hNGAL mutein binding pyoverdine (type I, II orIII) or pyochelin with detectable affinity may include at least oneamino acid substitution of a native cysteine residue by another aminoacid, for example, a serine residue. In some other embodiments, a muteinbinding pyoverdine or pyochelin with detectable affinity may include oneor more non-native cysteine residues substituting one or more aminoacids of wild-type hNGAL. In a further particular embodiment, an hNGALmutein according to the disclosure includes at least two amino acidsubstitutions of a native amino acid by a cysteine residue, hereby toform one or more cysteine briges. In some embodiments, said cysteinebridge may connect at least two loop regions. The definition of theseregions is used herein in accordance with Flower (Flower, 1996, supra,Flower, et al., 2000, supra) and Breustedt et al. (2005, supra).

In some embodiments, an hNGAL mutein of the disclosure does not bind toenterobactin.

In one aspect, the present disclosure includes various hNGAL muteinsthat bind pyoverdine or pyochelin with at least detectable affinity. Inthis sense, pyoverdine or pyochelin is regarded as a non-natural ligandof the reference wild-type hNGAL, where “non-natural ligand” refers to acompound that does not bind to wild-type human lipocalin 2 underphysiological conditions. By engineering wild-type hNGAL with one ormore mutations at certain sequence positions, the present inventors havedemonstrated that high affinity and high specificity for the non-naturalligand, pyoverdine or pyochelin, is possible. In some embodiments, at 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplet(s)encoding certain sequence positions on wild-type I human lipocalin 2, arandom mutagenesis may be carried out through substitution at thesepositions by a subset of nucleotide triplets.

Further, the muteins of the disclosure may have a mutated amino acidresidue at any one or more, including at least at any one, two, three,four, five, six, seven, eight, nine, ten, eleven or twelve, of thesequence positions corresponding to certain sequence positions of thelinear polypeptide sequence of hNGAL, such as sequence positions 28, 34,36, 39-42, 44-47, 49, 52, 54-55, 65, 68, 70, 72-75, 77, 79-81, 87, 96,100, 103, 106, 108, 123, 125, 127, 132, 134, 141 and 145 of the linearpolypeptide sequence of human NGAL (SEQ ID NO: 1).

A mutein of the disclosure may include the wild type (natural) aminoacid sequence of the “parental” protein scaffold (such as hNGAL) outsidethe mutated amino acid sequence positions. In some embodiments, an hNGALmutein according to the disclosure may also carry one or more amino acidmutations at a sequence position/positions as long as such a mutationdoes, at least essentially not hamper or not interfere with the bindingactivity and the folding of the mutein. Such mutations can beaccomplished very easily on DNA level using established standard methods(Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Illustrative examples of alterations of the amino acid sequence areinsertions or deletions as well as amino acid substitutions.

Such substitutions may be conservative, i.e. an amino acid residue isreplaced with an amino acid residue of chemically similar properties, inparticular with regard to polarity as well as size. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. On the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of the human lipocalin 2 as long as thesedeletions or insertion result in a stable folded/functional mutein (forexample, hNGAL muteins with truncated N- and C-terminus). In suchmutein, for instance, one or more amino acid residues are added ordeleted at the N- or C-terminus of the polypeptide. Generally such amutein may have about at least 70%, including at least about 80%, suchas at least about 85% amino acid sequence identity, with the amino acidsequence of the mature hNGAL. As an illustrative example, the presentdisclosure also encompasses hNGAL muteins as defined above, in which thefour amino acid residues (G-N—I-K; positions 95-98; SEQ ID NO: 130) ofthe linear polypeptide sequence of the mature hNGAL have been deleted(e.g. SEQ ID NO: 46).

The amino acid sequence of an hNGAL mutein disclosed herein has a highsequence identity to the mature hNGAL (SEQ ID NO: 1) when compared tosequence identities with other lipocalins. In this general context, theamino acid sequence of a mutein of the disclosure is at leastsubstantially similar to the amino acid sequence of the naturalwild-type hNGAL, with the proviso that possibly there are gaps (asdefined below) in an alignment that are the result of additions ordeletions of amino acids. A respective sequence of a mutein of thedisclosure, being substantially similar to the sequences of the maturehNGAL, has, in some embodiments, at least 70% identity or sequencehomology, at least 75% identity or sequence homology, at least 80%identity or sequence homology, at least 82% identity or sequencehomology, at least 85% identity or sequence homology, at least 87%identity or sequence homology, or at least 90% identity or sequencehomology including at least 95% identity or sequence homology, to thesequence of the mature hNGAL, with the proviso that the altered positionor sequence is retained and that one or more gaps are possible.

As used herein, a mutein of the disclosure “specifically binds” a target(for example, pyoverdine or pyochelin) if it is able to discriminatebetween that target and one or more reference targets, since bindingspecificity is not an absolute, but a relative property. “Specificbinding” can be determined, for example, in accordance with Westernblots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptide scans.

In one embodiment, the muteins of the disclosure are fused at itsN-terminus and/or its C-terminus to a fusion partner which is a proteindomain that extends the serum half-life of the mutein. In furtherparticular embodiments, the protein domain is a Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding peptide, or an albumin bindingprotein.

In another embodiment, the muteins of the disclosure are conjugated to acompound that extends the serum half-life of the mutein. Morepreferably, the mutein is conjugated to a compound selected from thegroup consisting of a polyalkylene glycol molecule, a hydroethylstarch,an Fc part of an immunoglobulin, a CH3 domain of an immoglobulin, a CH4domain of an immunoglobulin, an albumin binding peptide, and an albuminbinding protein.

In yet another embodiment, the current disclosure relates to a nucleicacid molecule comprising a nucleotide sequence encoding a muteindisclosed herein. The disclosure encompasses a host cell containing saidnucleic acid molecule.

Muteins Specific for Pyoverdine.

Study of the P. aeruginosa isolates so far helped classify pyoverdineinto three different types (Meyer et al., Use of Siderophores to TypePseudomonads: The Three Pseudomonas Aeruginosa Pyoverdine Systems,Microbiology, 1997; vol. 143 no. 1 35-43). Roughly 42% of the P.aeruginosa isolates have a pyoverdine system identical to that of Pvdtype I, 42% of the P. aeruginosa isolates behave like Pvd type II, while16% of the P. aeruginosa isolates belong to Pvd type III, respectively(Cornelis et al., 1989a; Table 4). Each type has three members(subtypes) differening in the side chain which is succinyl, succinamidor α-ketoglutaryl, namely, Pvd type I succinyl, Pvd type I succinamid,Pvd type I α-ketoglutaryl, Pvd type II succinyl, Pvd type II succinamid,Pvd type II α-ketoglutaryl, Pvd type III succinyl, Pvd type IIIsuccinamid and Pvd type III α-ketoglutaryl.

To tackle P. aeruginosa producing different types of pyoverdine, thepresent disclosure provides hNGAL muteins directed against differenttypes of pyoverdine. The disclosure also provides useful applicationsfor such muteins, methods of making pyoverdine-binding hNGAL muteinsdescribed herein as well as compositions comprising such muteins.Pyoverdine-binding hNGAL muteins of the disclosure as well ascompositions thereof may be used in methods of detecting pyoverdine in asample or in methods of binding of pyoverdine in a subject. No suchhNGAL muteins having these features attendant to the uses provided bypresent disclosure have been previously described.

Pyoverdine did not bind to the natural wild-type hNGAL, while hNGAL'snatural ligand, enterobactin, docks into the calyx of hNGAL with highaffinity. Pyoverdine, therefore, is a virulence factor and a stealthsiderophore that evades hNGAL recognition, allowing P. aeruginosa toestablish infection (Peek et al., Pyoverdine, the Major Siderophore inPseudomonas aeruginosa, Evades NGAL Recognition, InterdisciplinaryPerspectives on Infectious Diseases, 2012).

Accordingly, it is an object of the present disclosure to providemuteins derived from human neutrophil gelatinase associated lipocalin(NGAL), also termed as human lipocalin 2, which muteins, in contrast tonature wild-type hNGAL, have high specificity for pyoverdine.

Exemplary Muteins Specific for Pyoverdine.

In one aspect, the present disclosure relates to novel, specific-bindinghuman lipocalin 2 (human Lcn2 or hNGAL) muteins specific for one type ofpyoverdine, such as Pvd type I, Pvd type II or Pvd type III.

One embodiment of the current disclosure relates to a mutein that iscapable of binding one type of pyoverdine with detectable affinity, suchas an affinity measured by a K_(D) of about 200 nM or lower, such asabout 150 nM or lower.

In one aspect, the current disclosure provides an hNGAL mutein that iscapable of binding Pvd type I complexed with iron with a K_(D) of about20 nM or lower, such as 15 nM or lower, for example, when measured byBiacore T200 instrument in an assay essentially described in Example 6.

In some further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding Pvd type I succinyl, Pvd type Isuccinamid and Pvd type I α-ketoglutaryl with and without complexediron, with an affinity measured by an IC50 value of about 200 nM orlower, for example, when measured in an ELISA assay essentiallydescribed in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptakemediated by pyoverdine type I succinyl with an IC50 value of about 150nM or lower in a competition ELISA format essentially described inExample 7.

In some embodiments, the mutein is capable of inhibiting bacterialgrowth of Pvd I strain in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide, wherein saidpolypeptide includes an hNGAL mutein, and said hNGAL in comparison withthe linear polypeptide sequence of the mature hNGAL, comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 36, 39-41, 46, 49, 52, 54-55, 59, 65, 68, 70, 72-75, 77, 79-81, 87,96, 100, 103, 106, 125, 127, 132, 134 and 136, and wherein saidpolypeptide binds Pvd type I, including Pvd type I succinyl, Pvd type Isuccinamid and Pvd type I α-ketoglutaryl.

In some embodiments, a Pvd-type-I-binding hNGAL mutein of the disclosureincludes, at any one or more of the sequence positions 36, 40-41, 49,52, 68, 70, 72-73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132 and 134of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1),one or more of the following mutated amino acid residues: Leu 36→Asn.Thr, Val, Trp or Phe; Ala 40→Gly, Asn, Thr or Phe; Ile 41→Arg, Ala, Thr,Phe or Trp; Gln 49→Ile, Leu, Vla, Ala or Pro; Tyr 52→Met, Trp or Pro;Ser 68→Asp, Vla or Glu; Leu 70→Gln, Trp, Asp or Thr; Arg 72→Trp, Ala,Ser, Leu, Pro or Glu; Lys 73→Asp, Leu, Ala, Glu or Asn; Asp 77→Arg, Leu,Tyr, Ser, Gln, Thr, Ile or Asn; Trp 79→Gln, Asp, Ser, Arg, Met or Glu;Arg 81→Gln, Gly, Ile, Glu, His or Asp; Asn 96→His, Ile, Gly, Tyr or Asp;Tyr 100→Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103→Lys, Pro, Gln, His, Asp,Tyr, Glu, Trp or Asn; Tyr 106→His, Gln or Phe; Lys 125→Arg, Ser, Trp,Tyr, Val or Gly; Ser 127→Trp, Asn, Ala, Thr, Tyr, His, Ile, Val or Asp;Tyr 132→Trp, Asn, Gly or Lys; and Lys 134→Asn, His, Trp, Gly, Gln orAsp. In some embodiments, an hNGAL mutein of the disclosure includes twoor more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more or allmutated amino acid residues at these sequence positions of the maturehNGAL.

Additionally, a Pvd-type-I-binding hNGAL mutein according to thedisclosure may also comprise the following substitution in comparisonwith the linear polypeptide sequence of the mature hNGAL: Gln 28→His;Lys 46→Glu; Thr 54→Vla or Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Asp orGln; Ile 80→Thr; Cys 87→Ser or Asn; and Thr 136→Ala.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to Pvd type I, includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

Gln 28→His; Leu 36→Asn; Ala 40→Gly; Ile 41→Trp; Gln 49→Ile; Tyr 52→Met;Ser 68→Val; Leu 70→Gln; Arg 72→Trp; Lys 73→Asp; Asp 77→Leu; Trp 79→Gln;Arg 81→Gln; Cys 87→Ser; Asn 96→His; Tyr 100→Lys; Leu 103→His; Tyr106→His; Lys 125→Arg; Ser 127→Trp; Tyr 132→Trp; Lys 134→Asp;

Gln 28 His; Leu 36→Thr; Ala 40→Gly; Ile 41→Phe; Gln 49→Leu; Tyr 52→Trp;Leu 70→Trp; Arg 72→Ala; Lys 73→Leu; Asp 77→Tyr; Trp 79→Asp; Arg 81→Gly;Cys 87→Ser; Asn 96→Ile; Tyr 100→Glu; Leu 103→His; Tyr 106→Gln; Lys125→Trp; Ser 127→Asn; Tyr 132→Asn; Lys 134→Gln;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Ser; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Lys; Tyr106→His; Lys 125→Tyr; Ser 127→Ala; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Phe; Ala 40→Asn; Ile 41→Arg; Gln 49→Pro; Tyr 52→Met:Ser 68→Asp; Leu 70→Thr; Arg 72→Glu; Lys 73→Ala; Asp 77→Arg; Trp 79→Arg;Arg 81→Ile; Cys 87→Ser; Asn 96→Tyr; Tyr 100→Lys; Leu 103→Pro; Tyr106→Phe; Lys 125→Ser; Ser 127→Thr; Tyr 132→Trp; Lys 134→Gly;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Val; Tyr 52→Met; Ser 68→Val;Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu;Cys 87→Ser; Asn 96→Asp; Tyr 100→Phe; Leu 103→Trp; Tyr 106→Gln; Lys125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Gln 28→His; Leu 36→Val; Ala 40→Phe; Ile 41→Phe; Gln 49→Ala; Tyr 52→Pro;Ser 68→Glu; Leu 70→Trp; Arg 72→Leu; Lys 73→Asn; Asp 77→Gln; Trp 79→Glu;Arg 81→His; Cys 87→Ser; Asn 96→Tyr; Leu 103→Tyr; Tyr 106→His; Lys125→Val; Ser 127→His; Tyr 132→Lys; Lys 134→Trp;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser Lys 73→Glu; Asp 77→Ser; Trp 79→Ser;Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ser; Leu103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Ile; Tyr 132→Gly; Lys134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Ser; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asp: Tyr106→His; Lys 125→Tyr; Ser 127→Val; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Thr; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Asp; Tyr 100→Asn; Leu 103→Glu; Tyr106→His; Lys 125→Tyr; Ser 127→-Asp; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Val; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asn; Tyr106→His; Lys 125→Tyr; Ser 127→Vla; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Leu; Tyr 52→Met; Ser 68→Val;Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu;Cys 87→Ser; Asn 96→Asp; Tyr 100→Ser; Leu 103→Trp; Tyr 106→Gln; Lys125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Thr 54→Val; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Lys 75→Glu;Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly;Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Thr; Tyr132→Gly; Lys 134→Asn;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Ser 68→Val; Leu 70→Asp; Arg 72→Glu;Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met; Ile 80→Thr;Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu 103→Trp; Tyr106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Leu 36→Trp; Asn 39→Asp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Thr 54→Val; Asn 65→Asp; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu;Lys 75→Glu; Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser;Asn 96→Gly; Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser127→Thr; Tyr 132→Gly; Lys 134→Asn; Thr 136→Ala;

Leu 36→Trp; Ala 40→Thr; Ile 41→Ala; Gln 49→Pro; Tyr 52→Pro; Thr 54→Val;Asn 65→Asp; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Lys 75→Glu;Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly;Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Thr; Tyr132→Gly; Lys 134→Asn; Thr 136→Ala;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Asp; Ser 68→Val; Leu 70→Asp;Arg 72→Glu; Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met;Ile 80→Thr; Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys134→His; or

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Gln; Ser 68→Val; Leu 70→Asp;Arg 72→Glu; Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met;Ile 80→Thr; Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys134→His.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 39-41, 46, 49, 52, 54-55, 59, 65, 68, 70, 72-75, 77,79-81, 87, 96, 100, 103, 106, 125, 127, 132, 134 and 136, an hNGALmutein of the disclosure may include the wild type (natural) amino acidsequence outside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the currentdisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-18 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-I-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 2-18.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 2-18, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said hNGAL mutein.

A Pvd-type-I-binding hNGAL mutein according to the present disclosurecan be obtained by means of mutagenesis of a naturally occurring form ofhuman lipocalin 2. In some embodiments of the mutagenesis, asubstitution (or replacement) is a conservative substitution.Nevertheless, any substitution—including non-conservative substitutionor one or more from the exemplary substitutions below—is envisaged aslong as the mutein retains its capability to bind to Pvd type I, and/orit has an identity to the then substituted sequence in that it is atleast 60%, such as at least 65%, at least 70%, at least 75%, at least80%, at least 85% or higher identity to the amino acid sequence of themature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

In another aspect, the current disclosure provides an hNGAL mutein thatbinds Pvd type II complexed with iron with a K_(D) of about 20 nM orlower, such as 5 nM or lower, for example, when measured by Biacore T200instrument in an assay essentially described in Example 6.

In some still further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding Pvd type II succinyl, Pvd type IIsuccinamid and Pvd type II α-ketoglutaryl with and without complexediron, with an affinity measured by an IC50 value of about 200 nM orlower, for example, when measured in an ELISA assay essentiallydescribed in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptakemediated by pyoverdine type II succinyl with an IC50 value of about 150nM or lower in a competition ELISA format essentially described inExample 7.

In some embodiments, the mutein is capable of inhibiting bacterialgrowth of Pvd II strain in an assay essentially described in Example 8.

In some other embodiments, the mutein is capable of inhibiting orlessening growth of P. aeruginosa stains expressing pyoverdine type IIin an assay essentially described in Example 10.

In this regard, the disclosure relates to a polypeptide, wherein saidpolypeptide includes an hNGAL mutein, and said hNGAL in comparison withthe linear polypeptide sequence of the mature hNGAL, comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 36, 40-41, 49, 52, 54, 65, 68, 70, 72-75, 77, 79, 81, 87, 96, 100,103, 106, 125, 127, 132 and 134, and wherein said polypeptide binds Pvdtype II.

In some embodiments, a Pvd-type-II-binding hNGAL mutein of thedisclosure includes, at any one or more of the sequence positions 36,40-41, 49, 52, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125,127, 132 and 134 of the linear polypeptide sequence of the mature hNGAL(SEQ ID NO: 1), one or more of the following mutated amino acidresidues: Leu 36→Asn, Ile or Val; Ala 40→Glu, Gly, Asn, Thr or His; Ile41→Arg, Val or Thr; Gln 49→Gly, Ala or Pro; Tyr 52→Asn, Gly, Trp or Pro;Ser 68→Asp, Arg or Glu; Leu 70→Arg or Trp; Arg 72→His, Ile, Ala, Ser orGly; Lys 73→Asn, Met, Pro, Phe, Gln or Arg; Asp 77→His, Ile, Met, Lys,Gly or Asn; Trp 79→Ser, Tyr, Ala, Asp, Phe or Trp; Arg 81→Glu, Ser, Tyror Asp; Asn 96→Met, Ile, Arg, Asp, Lys, Asn or Ala; Tyr 100→Lys, Glu,Asn, Ser, Phe or Tyr; Leu 103→Thr, Ile, Gln, Gly, Met, His, Trp or Val;Tyr 106→Met, Gln, Ala, Ile, Asn, Gly, Met or Phe; Lys 125→Ala, Ile orAsn; Ser 127→Lys, Arg, Ser, Met, Asp or Asn; Tyr 132→Met, Phe, Asn, Ala,Ile, Gly or Val; and Lys 134→Trp or Tyr. In some embodiments, an hNGALmutein of the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or even more or all mutated amino acid residues at thesesequence positions of the mature hNGAL.

Additionally, a Pvd-type-II-binding hNGAL mutein according to thedisclosure may also comprise the following substitution in comparisonwith the linear polypeptide sequence of the mature hNGAL: Gln 28→His;Thr 54→Ala; Asn 65→Asp or Gln and Cys 87→Ser.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to Pvd type II, includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Tyr 100→Asn; Leu 103→Gln; Tyr 106→Met; Ser127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn; Ser 68→Asp;Leu 70→Arg; Arg 72→Ile; Lys 73→Met; Asp 77→His; Trp 79→Tyr; Arg 81→Glu;Cys 87→Ser; Asn 96→Ile; Tyr 100→Asn; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Met; Lys 134→Trp;

Gln 28→His; Leu 36→Ile; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→Ala; Lys 73→Pro; Asp 77→Ile; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Met; Tyr 100→Ser; Leu 103→Gly; Tyr106→Ala; Lys 125→Lys; Tyr 132→Val; Lys 134→Trp;

Gln 28→His; Ala 40→Asn; Gln 49→Ala; Tyr 52→Pro; Ser 68→Glu; Leu 70→Arg;Arg 72→Ser; Lys 73→Gln; Asp 77→Met; Trp 79→Ala; Arg 81→Tyr; Cys 87→Ser;Asn 96→Arg; Tyr 100→Pro; Leu 103→Thr; Tyr 106→Ile; Lys 125→Lys; Ser127→Met; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Ala 40→His; Gln 49→Ala; Tyr 52→Pro; Ser 68→Glu; Leu 70→Asp;Arg 72→Gly; Lys 73→Arg; Asp 77→His; Trp 79→Trp; Arg 81→Glu; Cys 87→Ser;Asn 96→Arg; Tyr 100→Asp; Leu 103→Met; Tyr 106→Phe; Lys 125→Ala; Ser127→Asp; Tyr 132→Asn; Lys 134→Trp;

Gln 28→His; Leu 36→Asn; Ala 40→Gly; Ile 41→Arg; Gln 49→Pro; Tyr 52→Trp;Ser 68→Arg; Leu 70→Trp; Arg 72→Asn; Lys 73→Gln; Asp 77→Lys; Trp 79→Asp;Arg 81→Glu; Cys 87→Ser; Asn 96→Asp; Tyr 100→Thr; Leu 103→Trp; Tyr106→Asn; Lys 125→Asn; Ser 127→Met; Tyr 132→Ile; Lys 134→Tyr;

Gln 28→His; Leu 36→Vla; Ala 40→Thr; Ile 41→Thr; Gln 49→Gly; Tyr 52→Gly;Ser 68→Glu; Leu 70→Arg; Arg 72→Gly; Lys 73→Arg; Asp 77→Gly; Trp 79→Trp;Arg 81→Glu; Cys 87→Ser; Asn 96→Ala; Tyr 100→Trp; Leu 103→Ile; Tyr106→Gly; Lys 125→Lys; Ser 127→Asn; Tyr 132→Val; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu 103→Val; Tyr106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Leu 103→Gln; Tyr 106→Met; Ser 127→Lys; Tyr132→Val; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Asp 77→Asn; Trp 79→Phe; Arg 81→Glu;Cys 87→Ser; Asn 96→Lys; Tyr 100→His; Leu 103→Gln; Tyr 106→Met; Ser127→Lys; Tyr 132→Ala; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Gly; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Trp;Arg 81→Glu; Cys 87→Ser; Tyr 100→Asn; Leu 103→His; Tyr 106→Met; Ser127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Phe; Asp 77→His; Trp 79→Tyr;Arg 81→Asp; Cys 87→Ser; Leu 103→Met; Tyr 106→Gln; Lys 125→Ile; Ser127→Arg; Tyr 132→Ile; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His; Trp 79→Tyr;Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys 125→Ile; Ser127→Arg; Tyr 132→Ile; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn;Trp 79→Phe; Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu103→Val; Tyr 106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Asn 65→Gln; Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn;Trp 79→Phe; Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu103→Val; Tyr 106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Thr 54→Ala; Asn 65→Asp; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg;Asp 77→His; Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr106→Gln; Lys 125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Thr 54→Ala; Asn 65→Gln; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg;Asp 77→His; Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr106→Gln; Lys 125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp;

Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn; Thr 54→Ala;Asn 65→Asp; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His;Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp; or

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Asn 65→Gln; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His;Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 40-41, 49, 52, 54, 65, 68, 70, 72-75, 77, 79, 81, 87,96, 100, 103, 106, 125, 127, 132 and 134, an hNGAL mutein of thedisclosure may include the wild type (natural) amino acid sequenceoutside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the currentdisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-37 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-II-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 19-37.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 19-37, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said hNGAL mutein.

A Pvd-type-II-binding hNGAL mutein according to the present disclosurecan be obtained by means of mutagenesis of a naturally occurring form ofhuman lipocalin 2. In some embodiments of the mutagenesis, asubstitution (or replacement) is a conservative substitution.Nevertheless, any substitution—including non-conservative substitutionor one or more from the exemplary substitutions below—is envisaged aslong as the mutein retains its capability to bind to Pvd type I, and/orit has an identity to the then substituted sequence in that it is atleast 60%, such as at least 65%, at least 70%, at least 75%, at least80%, at least 85% or higher identity to the amino acid sequence of themature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

In still another aspect, the current disclosure provides an hNGAL muteinthat binds Pvd type III complexed with iron with a K_(D) of about 20 nMor lower, such as 10 nM or lower, for example, when measured by BiacoreT200 instrument in an assay essentially described in Example 6.

In some still further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding Pvd type III succinyl, Pvd type IIIsuccinamid and Pvd type II α-ketoglutaryl with and without complexediron, with an affinity measured by an IC50 value of about 200 nM orlower, for example, when measured in an ELISA assay essentiallydescribed in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptakemediated by pyoverdine type III with an IC50 value of about 150 nM orlower in a competition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterialgrowth of Pvd III strain in an assay essentially described in Example 8.

In this regard, the disclosure relates to a polypeptide, wherein saidpolypeptide includes an hNGAL mutein, and said hNGAL in comparison withthe linear polypeptide sequence of the mature hNGAL, comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 36, 40-42, 45-47, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96,100, 103, 105-106, 125, 127, 132, 134 and 145, and wherein saidpolypeptide binds Pvd type III.

In some embodiments, a Pvd-type-III-binding hNGAL mutein of thedisclosure includes, at any one or more of the sequence positions 36,40-41, 49, 52, 68, 70, 72-73, 77, 79, 81, 96, 100, 103, 106, 125, 127,132 and 134 of the linear polypeptide sequence of the mature hNGAL (SEQID NO: 1), one or more of the following mutated amino acid residues: Leu36→Phe or Glu; Ala 40→Trp, Leu or Arg; Ile 41→Met, Arg, Ala, Leu or Trp;Gln 49→His, Ile, Arg, Lys, Met or Pro; Tyr 52→Asn, Tyr, Arg, Ser or Met;Ser 68→Asp, Asn, Glu or Gln; Leu 70→Lys, Asn or Arg; Arg 72→Leu, Arg,Gln or Tyr; Lys 73→His, Leu, Ala, Pro, Gln or Tyr; Asp 77→Ala, Ile, Lys,Gln or Arg; Trp 79→Ser or Asp; Arg 81→His, Ala, Ser or Val; Asn 96→Met,Ile, Arg, Gly, Leu or Val; Tyr 100→Ala, Ile, Asn, Pro or Asp; Leu103→Gln, Gly, Phe or Pro; Tyr 106→Glu; Lys 125→Trp or Thr; Ser 127→Val,His, Ile, Phe or Ala; Tyr 132→Phe; and Lys 134→Trp, Gln or Glu. In someembodiments, an hNGAL mutein of the disclosure includes two or more,such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more or all mutatedamino acid residues at these sequence positions of the mature hNGAL.

Additionally, a Pvd-type-III-binding hNGAL mutein according to thedisclosure may also comprise the following substitution in comparisonwith the linear polypeptide sequence of the mature hNGAL: Gln 28→His;Leu 42→Arg; Asp 45→Gly; Lys 46→Arg; Asp 47→Asn; Asn 65→Asp; Cys 87→Ser;Ser 105→Pro and Thr 145→Pro.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to Pvd type III, includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

Gln 28→His; Leu 36→Phe: Ala 40→Trp; Ile 41→Met; Gln 49→His; Tyr 52→Asn;Ser 68→Glu; Leu 70→Lys; Arg 72→Gln; Lys 73→Ala; Asp 77→Ile; Trp 79→Ser;Arg 81→His; Cys 87→Ser; Asn 96→Ile; Tyr 100→Asn; Leu 103→Gly; Tyr106→Glu; Lys 125→Trp; Ser 127→His; Tyr 132→Phe; Lys 134→Gln;

Gln 28→His; Leu 36→Phe; Ala 40→Arg; Ile 41→Trp; Gln 49→Ile; Tyr 52→Tyr;Ser 68→Gln; Leu 70→Asn; Arg 72→Trp; Lys 73→Leu; Asp 77→Ala; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Arg; Tyr 100→Ile; Leu 103→Pro; Tyr106→Glu; Lys 125→Thr; Ser 127→Ile; Tyr 132→Phe; Lys 134→Glu;

Gln 28→His; Leu 36→Phe; Ala 40→Leu; Ile 41→Leu; Gln 49→Arg; Tyr 52→Arg;Ser 68→Asp; Leu 70→Arg; Arg 72→Leu; Lys 73→Tyr; Asp 77→Ile; Trp 79→Ser;Arg 81→Ala; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ala; Leu 103→Phe; Tyr106→Glu; Lys 125→Trp; Ser 127→Ala; Lys 134→Glu;

Gln 28→His; Leu 36→Phe; Ala 40→Trp; Ile 41→Arg; Gln 49→Pro; Tyr 52→Ser;Ser 68→Asn; Leu 70→Arg; Arg 72→Trp; Lys 73→Pro; Asp 77→Arg; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Met; Tyr 100→Pro; Leu 103→Gly; Tyr106→Glu; Lys 125→Trp; Ser 127→Phe; Tyr 132→Phe; Lys 134→Glu;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Lys; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Gln; Trp 79→Asp; Arg 81→Ala;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Gln; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Thr; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Arg; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Vla; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Lys; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Tyr; Asp 77→Gln; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Tyr 100→Glu; Leu 103→Gln; Tyr 106→Glu; Ser 127→Val;Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met;Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 47→Asn; Gln 49→Met;Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp; Thr145→Pro;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 45→Gly; Lys 46→Arg;Gln 49→Met; Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met;Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 47→Asn; Gln 49→Met;Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp; Thr 145→Pro;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 45→Gly; Lys 46→Arg;Gln 49→Met; Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→, Arg; Lys73→His; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr100→Asp; Leu 103→Gln: Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr132→Phe; Lys 134→Trp; or

Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met; Tyr 52→Met;Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 40-42, 45-47, 49, 52, 65, 68, 70, 72-73, 77, 79, 81,87, 96, 100, 103, 105-106, 125, 127, 132, 134 and 145, an hNGAL muteinof the disclosure may include the wild type (natural) amino acidsequence outside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the currentdisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 38-53 or a fragment or variant thereof.

The amino acid sequence of a Pvd-type-III-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 38-53.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 38-53, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said hNGAL mutein.

A Pvd-type-III-binding hNGAL mutein according to the present disclosurecan be obtained by means of mutagenesis of a naturally occurring form ofhuman lipocalin 2. In some embodiments of the mutagenesis, asubstitution (or replacement) is a conservative substitution.Nevertheless, any substitution—including non-conservative substitutionor one or more from the exemplary substitutions below—is envisaged aslong as the mutein retains its capability to bind to Pvd type I, and/orit has an identity to the then substituted sequence in that it is atleast 60%, such as at least 65%, at least 70%, at least 75%, at least80%, at least 85% or higher identity to the amino acid sequence of themature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

Applications of Muteins Specific for Pyoverdine

Pyoverdines are the main siderophores of pseudomonads such as P.aeruginosa. In vitro experiments indicated a potential role of the P.aeruginosa pyoverdine in iron release from ferritransferrin but theability of pyoverdine to compete for iron in vivo has only recently beendemonstrated (Meyer et al., 1996, Infection and Immunity, 64, p.518-523). It was observed using a burned-mouse model that the absence ofpyoverdine production in mutants raised from a virulent parental straincorrelated with a loss of virulence of these mutants and that virulencewas restored when the homologous pyoverdine originating from thewild-type strain was supplemented. Furthermore, supplementation with aheterologous pyoverdine did not restore the virulence of the lattermutants. Thus, a precise knowledge of the pyoverdine-mediated ironuptake system used by a given P. aeruginosa isolate during infectionappears a prerequisite for developing new ways of treatment of P.aeruginosa infections via bacterial iron metabolism, e.g., by blockingthe pyoverdine biosynthesis or the pyoverdine-mediated iron transport.

Numerous possible applications for the pyoverdine-binding muteins of thedisclosure, therefore, exist in medicine. In one further aspect, thedisclosure relates to the use of a pyoverdine-binding mutein disclosedherein for detecting pyoverdine (type I, II or III) in a sample as wellas a respective method of diagnosis.

The present disclosure also involves the use of one or morepyoverdine-binding muteins as described for complex formation withpyoverdine (type I, II or III).

Therefore, in another aspect of the disclosure, the disclosed muteinsare used for the detection of pyoverdine (type I, II or III). Such usemay include the steps of contacting one or more said muteins, undersuitable conditions, with a sample suspected of containing pyoverdine,thereby allowing formation of a complex between the muteins andpyoverdine (type I, II or III), and detecting the complex by a suitablesignal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is surface plasmon resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The pyoverdine-binding muteins disclosed herein may also be used for theseparation of pyoverdine (type I, II or III). Such use may include thesteps of contacting one or more said muteins, under suitable conditions,with a sample supposed to contain pyoverdine (type I, II and/or III),thereby allowing formation of a complex between the muteins andpyoverdine (type I, II or III), and separating the complex from thesample. In the use of the disclosed muteins for the detection ofpyoverdine as well as the separation of pyoverdine (type I, II or III),the muteins and/or pyoverdine or a domain or fragment thereof may beimmobilized on a suitable solid phase.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a pyoverdine-binding mutein according to thedisclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure encompasses the use of a pyoverdine-binding mutein of thedisclosure or a composition comprising such mutein for the binding ofpyoverdine (type I, II or III) in a subject and/or inhibiting orlessening growth of P. aeruginosa in a subject.

In still another aspect, the present disclosure features a method ofbinding pyoverdine (type I, II or III) in a subject, comprisingadministering to said subject an effective amount of one or morepyoverdine-binding muteins of the disclosure or of one or morecompositions comprising such muteins.

In still another aspect, the present disclosure involves a method forinhibiting or lessening growth of P. aeruginosa in a subject, comprisingadministering to said subject an effective amount of one or morepyoverdine-binding muteins of the disclosure or of one or morecompositions comprising such muteins.

Muteins Specific for Pyochelin

In addition, the present disclosure fulfills the need for alternativeinhibitors of pyochelin by providing hNGAL muteins that bind pyochelinand useful applications therefor.

Accordingly, the disclosure also provides methods of making and usingthe pyochelin-binding muteins described herein as well as compositionsthat may be used in methods of detecting pyochelin in a sample or inmethods of binding of pyochelin in a subject. No such hNGAL muteinshaving these features attendant to the uses provided by presentdisclosure have been previously described.

Exemplary Muteins Specific for Pyochelin

In one aspect, the present disclosure relates to an hNGAL mutein thatbinds pyochelin complexed with iron with a K_(D) of about 20 nM orlower, such as 1 nM or lower, for example, when measured by Biacore T200instrument in an assay essentially described in Example 6.

In some still further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding pyochelin with complexed iron, with anaffinity measured by an IC50 value of about 500 nM or lower, forexample, when measured in an ELISA assay essentially described inExample 5.

In some still further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding pyochelin without complexed iron, withan affinity measured by an IC50 value of about 200 nM or lower, forexample, when measured in an ELISA assay essentially described inExample 5.

In some still further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding pyochelin with and without complexediron, with an affinity measured by an IC50 value of about 200 nM orlower, for example, when measured in an ELISA assay essentiallydescribed in Example 5.

In some embodiments, the mutein is capable of inhibiting iron uptakemediated by pyochelin with an IC50 value of about 150 nM or lower in acompetition ELISA format essentially described in Example 7.

In some embodiments, the mutein is capable of inhibiting bacterialgrowth of Pvd I knock-out (ΔpvdA) in an assay essentially described inExample 8.

In this regard, the disclosure relates to a polypeptide, wherein saidpolypeptide includes an hNGAL mutein, and said hNGAL in comparison withthe linear polypeptide sequence of the mature hNGAL, comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 34, 36, 40-41, 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77, 79-81, 87,96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141, and whereinsaid polypeptide binds pyochelin.

In some embodiments, a pyochelin-binding hNGAL mutein of the disclosureincludes, at any one or more of the sequence positions 36, 40-41, 49,52, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and134 of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO:1), one or more of the following mutated amino acid residues: Leu36→His, Met or Val; Ala 40→Ile, Gln, Tyr or Phe; Ile 41→Leu, His or Trp;Gln 49→His, Arg, Ser or Ala; Tyr 52→Leu, Trp or Pro; Ser 68→Asp or His;Leu 70→Arg or Trp; Arg 72→His, Ile, Ala, Ser or Gly; Lys 73→Asn, Met,Pro, Phe, Gln or Arg; Asp 77→Arg, Thr, Pro or Asp; Trp 79→Ala, Arg, Lysor Asp; Arg 81→Thr, Ile or Trp; Asn 96→Met, Asn, Pro or Ala; Tyr100→Gly, His or Glu; Leu 103→Gly, Met, His or Gln; Tyr 106→Met, Gly, Argor Trp; Lys 125→Trp, Phe, Gly or Leu; Ser 127→Arg, Trp, Asp or Ile; Tyr132→Ala, Glu or Thr; and Lys 134→Leu, Val, Asn or Phe. In someembodiments, an hNGAL mutein of the disclosure includes two or more,such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more or all mutatedamino acid residues at these sequence positions of the mature hNGAL.

Additionally, a pyochelin-binding hNGAL mutein according to thedisclosure may also comprise the following substitution in comparisonwith the linear polypeptide sequence of the mature hNGAL: Gln 28→His;Val 34→Leu; Glu 44→Gly; Asp 45→Gly; Lys→Arg or Tyr; Asn 65→Asp; Ile80→Thr; Cys 87→Ser; Leu 94→Phe; Val 108→Ala; Phe 123→Ser and Thr141→Ala.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to pyochelin, includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

Gln 28→His; Ala 40→Ile; Ile 41→Leu; Gln 49→His; Tyr 52→Leu; Ser 68→His;Leu 70→Thr; Arg 72→Lys; Lys 73→Trp; Asp 77→Ile; Trp 79→Ser; Arg 81→His;Cys 87→Ser; Asn 96→Met; Tyr 100→Asn; Leu 103→His; Tyr 106→Met; Lys125→Trp; Ser 127→Asp; Tyr 132→Glu; Lys 134→Leu;

Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Gln 49→Arg; Tyr 52→Trp;Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile; Asp 77→His; Trp 79→Arg;Arg 81→Thr; Cys 87→Ser; Tyr 100→His; Leu 103→Gly; Tyr 106→Gly; Lys125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys 134→Phe;

Gln 28→His; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser; Tyr 52→Pro;Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala; Trp 79→Lys;Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly; Leu 103→Met; Tyr106→Trp; Lys 125→Gly; Ser 127→Trp; Tyr 132→Thru; Lys 134→Val;

Gln 28→His; Leu 36→Val; Ala 40→Tyr; Ile 41→Trp; Gln 49→Ala; Ser 68→Asp;Leu 70→Arg; Arg 72→Trp; Lys 73→Arg; Asp 77→Arg; Trp 79→Asp; Arg 81→Trp;Cys 87→Ser; Asn 96→Pro; Tyr 100→Glu; Leu 103→Gln; Tyr 106→Arg; Lys125→Leu; Ser 127→Arg; Tyr 132→Ala; Lys 134→Asn;

Gln 28→His; Vla 34→Leu; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser;Tyr 52→Pro; Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala;Trp 79→Lys; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly;Leu 103→Met; Tyr 106→Trp; Phe 123→Ser; Lys 125→Gly; Ser 127→Trp; Tyr132→Thru; Lys 134→Val; Thr 141→Ala;

Gln 28→His; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser; Tyr 52→Pro;Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala; Trp 79→Lys;Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly; Leu103→Met; Tyr 106→Trp; Phe 123→Ser; Lys 125→Gly; Ser 127→Trp; Tyr132→Thru; Lys 134→Val;

Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Asp 45→Gly; Lys 46→Arg;Gln 49→Arg; Tyr 52→Trp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile;Asp 77→Leu; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Tyr 100→His; Leu103→Gly; Tyr 106→Gly; Lys 125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys134→Phe; Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Glu 44→Gly; Lys46→Tyr;

Gln 49→Arg; Tyr 52→Trp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile:Lys 74→Glu; Asp 77→His; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Leu 94→Phe;Tyr 100→His; Leu 103→Gly; Tyr 106→Gly; Val 108→Ala; Lys 125→Phe; Ser127→Ile; Tyr 132→Ala; Lys 134→Phe; or

Leu 36→His; Ala 40→Gln; Ile 41→Trp; Asp 45→Gly; Lys 46→Arg; Gln 49→Arg;Tyr 52→Trp; Asn 65→Asp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile;Asp 77→Leu; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Tyr 100→His; Leu103→Gly; Tyr 106→Gly; Lys 125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys134→Phe.

In the residual region, i.e. the region differing from sequencepositions 28, 34, 36, 40-41, 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77,79-81, 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141, anhNGAL mutein of the disclosure may include the wild type (natural) aminoacid sequence outside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the currentdisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 54-63 or a fragment or variant thereof.

The amino acid sequence of a pyochelin-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 54-63.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 54-63, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said hNGAL mutein.

A pyochelin-binding hNGAL mutein according to the present disclosure canbe obtained by means of mutagenesis of a naturally occurring form ofhuman lipocalin 2. In some embodiments of the mutagenesis, asubstitution (or replacement) is a conservative substitution.Nevertheless, any substitution—including non-conservative substitutionor one or more from the exemplary substitutions below—is envisaged aslong as the mutein retains its capability to bind to Pvd type I, and/orit has an identity to the then substituted sequence in that it is atleast 60%, such as at least 65%, at least 70%, at least 75%, at least80%, at least 85% or higher identity to the amino acid sequence of themature human lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).

2. Applications of Muteins Specific for Pyochelin

Pyochelin (Pch) is one of the two major siderophores produced andsecreted by Pseudomonas aeruginosa to assimilate iron. It chelates ironin the extracellular medium and transports it into the cell via aspecific outer membrane transporter, FptA. Pch strongly chelatesdivalent metals such as Zn(II) (pZn=11.8 at p[H] 7.4) and Cu(II)(pCu=14.9 at p[H] 7.4) and forms predominantly 1:2 (M²⁺/Pch) complexes.Siderophores are not only devoted to iron(III) shuttling but most likelydisplay other specific biological roles in the subtle metals homeostasisin microorganisms.

Numerous possible applications for the muteins with binding-affinity forpyochelin of the disclosure, therefore, exist in medicine. In onefurther aspect, the disclosure relates to the use of such a muteindisclosed herein for detecting pyochelin in a sample as well as arespective method of diagnosis.

The present disclosure also involves the use of one or more muteins withbinding-affinity for pyochelin as described for complex formation withpyochelin.

Therefore, in another aspect of the disclosure, the disclosed muteinsare used for the detection of pyochelin. Such use may include the stepsof contacting one or more said muteins, under suitable conditions, witha sample suspected of containing pyochelin, thereby allowing formationof a complex between the muteins and pyochelin, and detecting thecomplex by a suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is surface plasmon resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein may also be used for the separation ofpyochelin. Such use may include the steps of contacting one or more saidmuteins, under suitable conditions, with a sample supposed to containpyochelin, thereby allowing formation of a complex between the muteinsand pyochelin, and separating the complex from the sample.

In the use of the disclosed muteins for the detection of pyochelin aswell as the separation of pyochelin, the muteins and/or pyochelin or adomain or fragment thereof may be immobilized on a suitable solid phase.

Accordingly, the presence or absence of a molecule such as pyochelin,e.g., in a sample, as well as its concentration or level may bedetermined.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a mutein with binding-affinity for pyochelinaccording to the disclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure encompasses the use of such a mutein of the disclosure or acomposition comprising such mutein for the binding of pyochelin in asubject and/or inhibiting or lessening growth of P. aeruginosa in asubject.

In still another aspect, the present disclosure features a method ofbinding pyochelin in a subject, comprising administering to said subjectan effective amount of one or more muteins with binding-affinity forpyochelin of the disclosure or of one or more compositions comprisingsuch a mutein.

In still another aspect, the present disclosure involves a method forinhibiting or lessening growth of P. aeruginosa in a subject, comprisingadministering to said subject an effective amount of one or more muteinswith binding-affinity for pyochelin of the disclosure or of one or morecompositions comprising such a mutein.

C. Compositions Comprising Pyoverdine-Binding Mutein and/orPyochelin-Binding Mutein and Combination of the Muteins

P. aeruginosa is a species of bacterium that is widely distributed inthe environment and is capable of causing very severe infections inpatients with predisposing conditions, such as cystic fibrosis. P.aeruginosa synthesizes two major siderophores, pyoverdine (Pvd) andpyochelin (Pch), to cover its needs in iron(III). The biofilm mode ofgrowth is believed to be critical for persistent P. aeruginosainfections (Costerton et al., 1999; Singh et al., 2000) and the dualexpression of Pvd and Pch genes is necessary for normal biofilmdevelopment (Banin et al., 2005).

Given that P. aeruginosa produces an impressive array of virulencefactors, all playing a role in its pathogenicity, a preferred strategyto efficiently inhibit P. aeruginosa virulence is to target severalvirulence factors.

To this aim, the present disclosure encompasses use of (i) a firstmutein or polypeptide thereof specific for pyoverdine type I, (ii) asecond mutein or polypeptide thereof specific for pyoverdine type II,(iii) a third mutein or polypeptide thereof specific for pyoverdine typeIII and/or (iv) a fourth mutein or polypeptide thereof specific forpyochelin for the binding of pyoverdine type I, II, III and/or pyochelinin a subject. Such use includes a step of administering to a subject aneffective amount of (i) a first mutein or polypeptide thereof specificfor pyoverdine type I, (ii) a second mutein or polypeptide thereofspecific for pyoverdine type II, (iii) a third mutein or polypeptidethereof specific for pyoverdine type III and/or (iv) a fourth mutein orpolypeptide thereof specific for pyochelin. The present disclosure alsocontemplates the use of (i) a first mutein or polypeptide thereofspecific for pyoverdine type I, (ii) a second mutein or polypeptidethereof specific for pyoverdine type II, (iii) a third mutein orpolypeptide thereof specific for pyoverdine type III and/or (iv) afourth mutein or polypeptide thereof specific for pyochelin forpreventing or reducing iron-uptake by P. aeruginosa through pyochelinand/or pyoverdine in a subject. Similarly, the present disclosurediscloses the use of (i) a first mutein or polypeptide thereof specificfor pyoverdine type I, (ii) a second mutein or polypeptide thereofspecific for pyoverdine type II, (iii) a third mutein or polypeptidethereof specific for pyoverdine type III and/or (iv) a fourth mutein orpolypeptide thereof specific for pyochelin for the treatment oralleviation of P. aeruginosa infection and/or biofilm formation in asubject. In some further embodiments, the P. aeruginosa infection can beacute or chronic infections.

The first, second, third and/or fourth muteins or polypeptides thereofmay be administered in combination, including concurrently,concomittantly or in series. In some embodiments, the first, second,third and/or fourth muteins or polypeptides thereof may be included in acomposition that may be administered. The composition may include aneffective amount of the first, second, third and/or fourth muteins orpolypeptides thereof as active ingredients, in association with at leastone pharmaceutically acceptable adjuvant, diluent or carrier. The first,second, third and/or fourth muteins or polypeptides thereof may also beadministered independent from each other, including at individualintervals at independent points of time.

In some embodiments, the mutein specific for pyoverdine (type I, II orIII) as used in the disclosure is able to bind pyoverdine (type I, II orIII, respectively) with detectable affinity, i.e. with a dissociationconstant of at least 200 nM, including about 100 nM, about 50 nM, about25 nM or about 15 nM. In some embodiments, the mutein specific forpyochelin as used in the disclosure is able to bind pyochelin withdetectable affinity, i.e. with a dissociation constant of at least 200nM including about 100 nM, about 50 nM, about 25 nM or about 15 nM. Insome further preferred embodiments, a mutein of the combinationaccording to the disclosure binds pyoverdine (type I, II or III) orpyochelin, respectively, with a dissociation constant for pyoverdine(type I, II or III, respectively) or pyochelin of at least about 10 nM,about 1 nM, about 0.1 nM, about 10 μM, or even lower. The presentdisclosure, thus, provides a combination of (i) a mutein of hNGAL thathas a detectable affinity to pyoverdine type I (Pvd I s, sa, aKG+/−Fe),(ii) a mutein of hNGAL that has a detectable affinity to pyoverdine typeII (Pvd II s, sa, aKG+/−Fe), (iii) a mutein of hNGAL that has adetectable affinity to pyoverdine type III (Pvd III s, sa, aKG+/−Fe)and/or (iv) a mutein of hNGAL that has a detectable affinity topyochelin (Pch+/−Fe).

Further details on hNGAL muteins with a detectable affinity forpyoverdine can be found in Section A of the current disclosure.

In a particularly preferred embodiment, a mutein that is specific forpyoverdine type I is shown in any one of SEQ ID NOs: 2-18. In aparticularly preferred embodiment, a mutein that is specific forpyoverdine type II is shown in any one of SEQ ID NOs: 19-37.

In a particularly preferred embodiment, a mutein that is specific forpyoverdine type III is shown in any one of SEQ ID NOs: 38-53.

Further details of hNGAL muteins with a detectable affinity forpyochelin have been disclosed in Section B of the current disclosure.

In a particular preferred embodiment, the mutein that is specific forpyochelin is shown in any one of SEQ ID NOs: 54-63.

The present disclosure also relates to a composition comprising at leastone of the following: (i) a first mutein or polypeptide thereof specificfor pyoverdine type I, (ii) a second mutein or polypeptide thereofspecific for pyoverdine type II, (iii) a third mutein or polypeptidethereof specific for pyoverdine type III and (iv) a fourth mutein orpolypeptide thereof specific for pyochelin, which composition can beused in a method of binding of pyoverdine type I, II, III and/orpyochelin.

The present disclosure relates to a combination of a first mutein orpolypeptide or composition thereof, a second mutein or polypeptide orcomposition thereof, a third mutein or polypeptide or compositionthereof, and/or a fourth mutein or polypeptide or composition thereof.One of these muteins can bind to pyoverdine (type I, II or III) as agiven non-natural target with detectable affinity. One of these muteinscan bind to pyochelin as a given non-natural target with detectableaffinity. The respective mutein thus binds to pyoverdine type I, II, IIIor pyochelin, respectively, as a given non-natural target. The term“non-natural target” refers to a compound, which does not bind to thecorresponding lipocalin (the wild-type hNGAL) under physiologicalconditions. For example, the first mutein or polypeptide or compositionthereof can bind to one type of pyoverdine (type I, II or III) orpyochelin and the second, the third or the fourth mutein or polypeptideor composition thereof can bind to pyochelin or an another type ofpyoverdine respectively, or vice versa. The combination of the first,the second, the third and/or the fourth muteins or polypeptides orcompositions thereof may be provided in various forms and orientations.

In still another aspect, the present disclosure features a method ofbinding pyoverdine type I, II, III and/or pyochelin in a subjectcomprising administering to said subject an effective amount of acomposition that comprises at least one of the following: (i) a muteinor polypeptide thereof specific for pyoverdine type I, (ii) a mutein orpolypeptide thereof specific for pyoverdine type II, (iii) a mutein orpolypeptide thereof specific for pyoverdine type III and (iv) a muteinor polypeptide thereof specific for pyochelin. In some embodiments, suchcomposition comprises two or more of, e.g. three or even all of(i)-(iv).

In still another aspect, the present disclosure involves a method forinhibiting or lessening growth of P. aeruginosa in a subject comprisingadministering to said subject an effective amount of a composition thatcomprises at least one of the following: (i) a mutein or polypeptidethereof specific for pyoverdine type I. (ii) a mutein or polypeptidethereof specific for pyoverdine type II, (iii) a mutein or polypeptidethereof specific for pyoverdine type III and (iv) a mutein orpolypeptide thereof specific for pyochelin. In some embodiments, suchcomposition comprises two or more of, e.g. three or even all of(i)-(iv).

The present disclosure also involves the use of (i) a first mutein orpolypeptide thereof specific for pyoverdine type I, (ii) a second muteinor polypeptide thereof specific for pyoverdine type II, (iii) a thirdmutein or polypeptide thereof specific for pyoverdine type III, and/or(iv) a fourth mutein or polypeptide thereof specific for pyochelin, forcomplex formation with pyoverdine type I, II, III and/or pyochelin.

Therefore, in another aspect of the disclosure, the disclosed muteins orpolypeptides can be used for the detection of pyoverdine and pyochelin.Such use may include the steps of contacting one or more said muteins orpolypeptides, under suitable conditions, with a sample suspected ofcontaining pyoverdine and/or pyochelin, thereby allowing formation of acomplex between the muteins or polypeptides and pyoverdine and/orbetween the muteins and pyochelin, respectively, and detecting thecomplex by a suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is surface plasmon resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins or polypeptides disclosed herein may also be used for theseparation of pyoverdine and/or pyochelin. Such use may include thesteps of contacting one or more said muteins, under suitable conditions,with a sample supposed to contain pyoverdine and/or pyochelin, therebyallowing formation of a complex between the muteins and pyoverdineand/or between the muteins and pyochelin, respectively, and separatingthe complex from the sample.

In the use of the disclosed muteins or polypeptides for the detection ofpyoverdine and/or pyochelin as well as the separation of pyoverdineand/or pyochelin, the muteins and/or pyoverdine and pyochelin or adomain or fragment thereof may be immobilized on a suitable solid phase.

Accordingly, the presence or absence of pyoverdine and/or pyochelin,e.g., in a sample, as well as its concentration or level may bedetermined.

In another aspect, the disclosure provides for a kit of parts. The kitincludes, in one or more containers, separately or in a mixture, amutein or polypeptide specific for pyoverdine type I or compositionthereof, a mutein or polypeptide specific for pyoverdine type II orcomposition thereof, a mutein or polypeptide specific for pyoverdinetype III or composition thereof, and/or a mutein or polypeptide specificfor pyochelin or composition thereof. In some further preferredembodiments, the kit comprises a first container that includes a firstmutein or polypeptide specific for pyoverdine type I or compositionthereof, a second container that includes a second mutein or polypeptidespecific for pyoverdine type II or composition thereof, a thirdcontainer that includes a third mutein or polypeptide specific forpyoverdine type III or composition thereof, and/or a fourth containerthat includes a fourth mutein or polypeptide specific for pyochelin orcomposition thereof. In some embodiments the kit further includesintegrally thereto or as one or more separate documents, informationpertaining to the contents or the kit and the use of the muteins orpolypeptides thereof. The kit may include in some embodiments one ormore compositions that are formulated for reconstitution in a diluent.Such a diluent, e.g. a sterile diluent, may also be included in the kit,for example within a container.

D. Muteins of the Disclosure

When used herein in the context of the muteins of the present disclosurethat bind to pyoverdine or pyochelin, the term “specific for” includesthat the mutein is directed against, binds to, or reacts with pyoverdineor pyochelin, respectively. Thus, being directed to, binding to orreacting with includes that the mutein specifically binds to pyoverdineor pyochelin, respectively. The term “specifically” in this contextmeans that the mutein reacts with a pyoverdine protein or a pyochelinprotein, as described herein, but essentially not with another protein.The term “another protein” includes any non-pyoverdine or non-pyochelinprotein, respectively, including proteins closely related to or beinghomologous to pyoverdine or pyochelin against which the muteinsdisclosed herein are directed to. However, pyoverdine or pyochelinproteins, fragments and/or variants from species other than human suchas those described in the context of the definition “subject” are notexcluded by the term “another protein”. The term “does not essentiallybind” means that the mutein of the present disclosure does not bindanother protein, i.e., shows a cross-reactivity of less than 30%,preferably 20%, more preferably 10%, particularly preferably less than9, 8, 7, 6 or 5%. Whether the mutein specifically reacts as definedherein above can easily be tested, inter alia, by comparing the reactionof a lipoclin mutein of the present disclosure with pyoverdine orpyochelin and the reaction of said mutein with (an) other protein(s).“Specific binding” can also be determined, for example, in accordancewith Western blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC andpeptide scans.

The amino acid sequence of a mutein according to the disclosure has ahigh sequence identity to human lipocalin 2 when compared to sequenceidentities with another lipocalin (see also above). In this generalcontext the amino acid sequence of a mutein of the combination accordingto the disclosure is at least substantially similar to the amino acidsequence of the corresponding lipocalin (the wild-type hNGAL). Arespective sequence of a mutein of the combination according to thedisclosure, being substantially similar to the sequence of mature hNGAL,such as at at least 65%, at least 70%, at least 75%, at least 80%, atleast 82%, at least 85%, at least 87%, at least 90% identity, includingat least 95% identity to the sequence of mature hNGAL. In this regard, amutein of the disclosure of course may contain, in comparisonsubstitutions as described herein which renders the mutein capable ofbinding to pyoverdine type I, II, III or pyochelin, respectively.Typically a mutein of hNGAL includes one or more mutations—relative tothe native sequence of hNGAL—of amino acids in the four loops at theopen end of the ligand binding site of hNGAL. As explained above, theseregions are essential in determining the binding specificity of a muteinfor pyoverdine type I, II, III or pyochelin. A mutein derived hNGAL or ahomologue thereof, may have one, two, three, four or more mutated aminoacid residues at any sequence position in the N-terminal region and/orin the three peptide loops BC, DE, and FG arranged at the end of theβ-barrel structure that is located opposite to the natural bindingpocket.

A mutein according to the disclosure includes one or more, such as two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen oreven twenty substitutions in comparison to the corresponding nativehNGAL alin, provided that such a mutein should be capable of binding topyoverdine or pyochelin, respectively. For example, a mutein can have asubstitution at a position corresponding to a distinct position (i.e. ata corresponding position) of hNGAL. In some embodiments a mutein of thecombination according to the disclosure includes at least two amino acidsubstitutions, including 2, 3, 4, 5, ors even more, amino acidsubstitutions of a native amino acid by an arginine residue.

Accordingly, the nucleic acid of a protein ‘reference’ scaffold asdescribed herein is subject to mutagenesis with the aim of generating amutein which is capable of binding to pyoverdine type I, II, III orpyochelin, respectively.

Also, a mutein of the present disclosure can comprise a heterologousamino acid sequence at its N- or C-Terminus, preferably C-terminus, suchas a Strep-tag, e.g., Strep II tag without affecting the biologicalactivity (binding to its target e.g. pyoverdine or pyochelin,respectively) of the mutein.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a mutein different from wild-type hNGALcorresponds to a certain position in the amino acid sequence ofwild-type hNGAL, a skilled artisan can use means and methods well-knownin the art, e.g., alignments, either manually or by using computerprograms such as BLAST2.0, which stands for Basic Local Alignment SearchTool or ClustalW or any other suitable program which is suitable togenerate sequence alignments. Accordingly, wild-type hNGAL can serve as“subject sequence” or “reference sequence”, while the amino acidsequence of a mutein different from the wild-type hNGAL described hereinserves as “query sequence”. The terms “reference sequence” and “wildtype sequence” are used interchangeably herein.

In some embodiments a substitution (or replacement) is a conservativesubstitution. Nevertheless, any substitution—including non-conservativesubstitution or one or more from the exemplary substitutions listedbelow—is envisaged as long as the mutein retains its capability to bindto pyoverdine type I, II, III or pyochelin, respectively, and/or it hasan identity to the then substituted sequence in that it is at least 60%,such as at least 65%, at least 70%, at least 75%, at least 80%, at least85% or higher identical to the “original” sequence.

Conservative substitutions are generally the following substitutions,listed according to the amino acid to be mutated, each followed by oneor more replacement(s) that can be taken to be conservative: Ala→Gly,Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp;Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln,Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr;Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also permissible andcan be determined empirically or in accord with other known conservativeor non-conservative substitutions. As a further orientation, thefollowing eight groups each contain amino acids that can typically betaken to define conservative substitutions for one another:

Alanine (Ala), Glycine (Gly);

Aspartic acid (Asp), Glutamic acid (Glu);

Asparagine (Asn), Glutamine (Gln);

Arginine (Arg), Lysine (Lys);

Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val);

Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);

Serine (Ser), Threonine (Thr); and

Cysteine (Cys), Methionine (Met)

If such substitutions result in a change in biological activity, thenmore substantial changes, such as the following, or as further describedbelow in reference to amino acid classes, may be introduced and theproducts screened for a desired characteristic.

Examples of such more substantial changes are: Ala→Leu, Ile; Arg→Gln;Asn→Asp, Lys, Arg, His; Asp→Asn; Cys→Ala; Gln→Glu; Glu→Gln; His→Lys;Ile→Met, Ala, Phe; Leu→Ala, Met, Norleucine; Lys→Asn; Met→Phe; Phe→Val,Ile, Ala; Trp→Phe; Tyr→Thr, Ser; Val→Met, Phe, Ala.

Substantial modifications in the biological properties of hNGAL areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, methionine, alanine, valine,leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine,threonine; (3) acidic: asparitic acid, glutamic acid; (4) basic:asparagine, glutamine, histidine, lysine, arginine; (5) residues thatinfluence chain orientation: glycine, proline; and (6) aromatic:tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of hNGAL also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to improve its stability.

Any mutation, including an insertion as discussed above, can beaccomplished very easily on the nucleic acid, e.g. DNA level usingestablished standard methods.

Illustrative examples of alterations of the amino acid sequence areinsertions or deletions as well as amino acid substitutions. Suchsubstitutions may be conservative, i.e. an amino acid residue isreplaced with an amino acid residue of chemically similar properties, inparticular with regard to polarity as well as size. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) iso-leucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. On the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of hNGAL as long as these deletions orinsertion result in a stable folded/functional mutein.

Modifications of the amino acid sequence include directed mutagenesis ofsingle amino acid positions in order to simplify sub-cloning of themutated hNGAL gene or its parts by incorporating cleavage sites forcertain restriction enzymes. In addition, these mutations can also beincorporated to further improve the affinity of a mutein for a giventarget such as pyoverdine or pyochelin. Furthermore, mutations can beintroduced in order to modulate certain characteristics of the muteinsuch as to improve folding stability, serum stability, proteinresistance or water solubility or to reduce aggregation tendency, ifnecessary. For example, naturally occurring cysteine residues may bemutated to other amino acids to prevent disulphide bridge formation. Itis also possible to deliberately mutate other amino acid sequenceposition to cysteine in order to introduce new reactive groups, forexample for the conjugation to other compounds, such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins,or for the formation of non-naturally occurring disulphide linkages. Thegenerated thiol moiety may be used to PEGylate or HESylate the mutein,for example, in order to increase the serum half-life of a respectivemutein.

It is also possible to mutate other amino acid sequence positions tocysteine in order to introduce new reactive groups, for example, for theconjugation to other compounds, such as polyethylene glycol (PEG),hydroxyethyl starch (HES), biotin, peptides or proteins, or for theformation of non-naturally occurring disulphide linkages.

In some embodiments, if one of the above moieties is conjugated to amutein of the disclosure, conjugation to an amino acid side chain can beadvantageous. Suitable amino acid side chains may occur naturally in theamino acid sequence of hNGAL or may be introduced by mutagenesis. Incase a suitable binding site is introduced via mutagenesis, onepossibility is the replacement of an amino acid at the appropriateposition by a cysteine residue.

With respect to a mutein of human lipocalin 2, exemplary possibilitiesof such a mutation to introduce a cysteine residue into the amino acidsequence of a lipocalin including human lipocalin 2 mutein to includethe introduction of a cysteine (Cys) residue at at least at one of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of humanNGAL. In some embodiments where a human lipocalin 2 mutein of thedisclosure has a sequence in which, in comparison to the sequence of theSWISS-PROT/UniProt Data Bank Accession Number P80188, a cysteine hasbeen replaced by another amino acid residue, the corresponding cysteinemay be reintroduced into the sequence. As an illustrative example, acysteine residue at amino acid position 87 may be introduced in such acase by reverting to a cysteine as originally present in the sequence ofSWISS-PROT accession No P80188. The generated thiol moiety at the sideof any of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145,143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, forexample, in order to increase the serum half-life of a respective humanlipocalin 2 mutein.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above compounds to a mutein accordingto the present disclosure, artificial amino acids may be introduced bymutagenesis. Generally, such artificial amino acids are designed to bemore reactive and thus to facilitate the conjugation to the desiredcompound. One example of such an artificial amino acid that may beintroduced via an artificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins disclosed herein it may beadvantageous to use them in the form of fusion proteins. In someembodiments, a mutein of the disclosure is fused at its N-terminus orits C-terminus to a protein, a protein domain or a peptide, forinstance, a signal sequence and/or an affinity tag.

Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of suitable fusion partners. Finally, proteins withchromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for muteins of the disclosure as well.

In general, it is possible to label the muteins of the disclosure withany appropriate chemical substance or enzyme, which directly orindirectly generates a detectable compound or signal in a chemical,physical, optical, or enzymatic reaction. An example for a physicalreaction and at the same time optical reaction/marker is the emission offluorescence upon irradiation or the emission of X-rays when using aradioactive label.

Alkaline phosphatase, horseradish peroxidase and β-galactosidase areexamples of enzyme labels (and at the same time optical labels) whichcatalyze the formation of chromogenic reaction products. In general, alllabels commonly used for antibodies (except those exclusively used withthe sugar moiety in the Fc part of immunoglobulins) can also be used forconjugation to the muteins of the disclosure. The muteins of thedisclosure may also be conjugated with any suitable therapeuticallyactive agent, e.g., for the targeted delivery of such agents to a givencell, tissue or organ or for the selective targeting of cells, e.g., oftumor cells without affecting the surrounding normal cells. Examples ofsuch therapeutically active agents include radionuclides, toxins, smallorganic molecules, and therapeutic peptides (such as peptides acting asagonists/antagonists of a cell surface receptor or peptides competingfor a protein binding site on a given cellular target). The muteins ofthe disclosure may, however, also be conjugated with therapeuticallyactive nucleic acids such as antisense nucleic acid molecules, smallinterfering RNAs, micro RNAs or ribozymes. Such conjugates can beproduced by methods well known in the art.

As indicated above, a mutein of the disclosure may in some embodimentsbe conjugated to a moiety that extends the serum half-life of the mutein(in this regard see also PCT publication WO 2006/56464 where suchconjugation strategies are described with references to muteins of humanneutrophile gelatinase-associated lipocalin with binding affinity forCTLA-4). The moiety that extends the serum half-life may be apolyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules,such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9),an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, aCH4 domain of an immunoglobulin, an albumin binding peptide, or analbumin binding protein, transferrin to name only a few. The albuminbinding protein may be a bacterial albumin binding protein, an antibody,an antibody fragment including domain antibodies (see U.S. Pat. No.6,696,245, for example), or a mutein with binding activity for albumin.Accordingly, suitable conjugation partners for extending the half-lifeof a mutein of the disclosure include an albumin binding protein, forexample, a bacterial albumin binding domain, such as the one ofstreptococcal protein G (König, T., & Skerra, A. (1998) J. Immunol.Methods 218, 73-83). Other examples of albumin binding peptides that canbe used as conjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al. (SEQID NO: 131; Dennis, M. S., Zhang, M., Meng, Y. G., Kadkhodayan, M.,Kirchhofer, D., Combs, D. & Damico, L. A. (2002) J Biol Chem 277,35035-35043).

In other embodiments, albumin itself (Osborn, B. L. et al., 2002, J.Pharmacol. Exp. Ther. 303, 540-548), or a biological active fragment ofalbumin can be used as conjugation partner of a mutein of thedisclosure. The term “albumin” includes all mammal albumins such ashuman serum albumin or bovine serum albumin or rat albumine. The albuminor fragment thereof can be recombinantly produced as described in U.S.Pat. No. 5,728,553 or European patent applications EP 0 330 451 and EP 0361 991. Recombinant human albumin (Recombumin®) Novozymes Delta Ltd.(Nottingham, UK) can be conjugated or fused to a mutein of thedisclosure in order to extend the half-life of the mutein.

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and MA, USA).

Using transferrin as a moiety to extend the serum half-life of themuteins of the disclosure, the muteins can be genetically fused to the Nor C terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer/half-life extension partner is alsocommercially available from Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the muteins of the disclosure, the SynFusion™technology, commercially available from Syntonix Pharmaceuticals, Inc(MA. USA), may be used. The use of this Fc-fusion technology allows thecreation of longer-acting biopharmaceuticals and may for example consistof two copies of the mutein linked to the Fc region of an antibody toimprove pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of the muteins of thedisclosure is to fuse to the N- or C-terminus of the muteins long,unstructured, flexible glycine-rich sequences (for example poly-glycinewith about 20 to 80 consecutive glycine residues). This approachdisclosed in WO2007/038619, for example, has also been term “rPEG”(recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkyleneglycol can be substituted, unsubstituted, linear or branched. It canalso be an activated polyalkylene derivative. Examples of suitablecompounds are polyethylene glycol (PEG) molecules as described in WO99/64016, in U.S. Pat. No. 6,177,074 or in U.S. Pat. No. 6,403,564 inrelation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (see for example, Fuertges et al. (1990) TheClinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins J. Control.Release 11, 139-148). The molecular weight of such a polymer, such aspolyethylene glycol, may range from about 300 to about 70.000 Dalton,including, for example, polyethylene glycol with a molecular weight ofabout 10.000, of about 20.000, of about 30.000 or of about 40.000Dalton. Moreover, as e.g. described in U.S. Pat. No. 6,500,930 or6,620,413, carbohydrate oligo- and polymers such as starch orhydroxyethyl starch (HES) can be conjugated to a mutein of thedisclosure for the purpose of serum half-life extension.

In addition, a mutein disclosed herein may be fused to a moiety mayconfer new characteristics to the muteins of the disclosure such asenzymatic activity or binding affinity for other molecules. Examples ofsuitable fusion partners are alkaline phosphatase, horseradishperoxidase, gluthation-S-transferase, the albumin-binding domain ofprotein G, protein A, antibody fragments, oligomerization domains ortoxins.

In particular, it may be possible to fuse a mutein disclosed herein witha separate enzyme active site such that both “components” of theresulting fusion protein together act on a given therapeutic target. Thebinding domain of the mutein attaches to the disease-causing target,allowing the enzyme domain to abolish the biological function of thetarget.

The present disclosure also relates to nucleic acid molecules (DNA andRNA) that include nucleotide sequences encoding the muteins of thedisclosure. Since the degeneracy of the genetic code permitssubstitutions of certain codons by other codons specifying the sameamino acid, the disclosure is not limited to a specific nucleic acidmolecule encoding a mutein as described herein but encompasses allnucleic acid molecules that include nucleotide sequences encoding afunctional mutein. In this regard, the present disclosure providesnucleotide sequences encoding some muteins of the disclosure as shown inSEQ ID NOs: 65-126.

In one embodiment of the disclosure, the method includes subjecting thenucleic acid molecule to mutagenesis at nucleotide triplets coding forat least one, or even more, of the sequence positions corresponding tothe sequence positions 28, 34, 36, 39-42, 44-47, 49, 52, 54-55, 65, 68,70, 72-75, 77, 79-81, 87, 96, 100, 103, 106, 108, 123, 125, 127, 132,134, 141 and 145 of the linear polypeptide sequence of human NGAL (SEQID NO: 2).

The disclosure also includes nucleic acid molecules encoding the muteinsof the disclosure, which include additional mutations outside theindicated sequence positions of experimental mutagenesis. Such mutationsare often tolerated or can even prove to be advantageous, for example ifthey contribute to an improved folding efficiency, serum stability,thermal stability or ligand binding affinity of the muteins.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it includes sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgamo element in prokaryotes or theTATA box, CAAT sequences, and 5′-capping elements in eukaryotes. Theseregions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, such as a promoter sequence. In some embodiments anucleic acid molecule of the disclosure includes a promoter sequence anda transcriptional termination sequence.

Suitable prokaryotic promoters are, for example, the tet promoter, thelacUV5 promoter or the T7 promoter. Examples of promoters useful forexpression in eukaryotic cells are the SV40 promoter or the CMVpromoter.

The nucleic acid molecules of the disclosure can also be part of avector or any other kind of cloning vehicle, such as a plasmid, aphagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is included in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (see e.g.Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a mutein asdescribed herein, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding a mutein as described herein, and inparticular a cloning vector containing the coding sequence of such amutein can be transformed into a host cell capable of expressing thegene. Transformation can be performed using standard techniques. Thus,the disclosure is also directed to a host cell containing a nucleic acidmolecule as disclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of thedisclosure. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) orprimary mammalian cells.

The disclosure also relates to a method for the production of a muteinor a polypeptide thereof as described herein, wherein the mutein orpolypeptide, a fragment of the mutein or polypeptide or a fusion proteinof the mutein or polypeptide and another polypeptide is producedstarting from the nucleic acid coding for the mutein or polypeptide bymeans of genetic engineering methods. The method can be carried out invivo, the mutein or polypeptide can for example be produced in abacterial or eucaryotic host organism and then isolated from this hostorganism or its culture. It is also possible to produce a protein invitro, for example by use of an in vitro translation system.

When producing the mutein or polypeptide thereof in vivo a nucleic acidencoding such mutein or polypeptide is introduced into a suitablebacterial or eukaryotic host organism by means of recombinant DNAtechnology (as already outlined above). For this purpose, the host cellis first transformed with a cloning vector that includes a nucleic acidmolecule encoding a mutein as described herein using establishedstandard methods. The host cell is then cultured under conditions, whichallow expression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide.

Subsequently, the polypeptide is recovered either from the cell or fromthe cultivation medium.

In some embodiments, a nucleic acid molecule, such as DNA, disclosed inthis application may be “operably linked” to another nucleic acidmolecule of the disclosure to allow expression of a fusion proteion ofthe disclosure. In this regard, an operable linkage is a linkage inwhich the sequence elements of the first nucleic acid molecule and thesequence elements of the second nucleic acid molecule are connected in away that enables expression of the fusion protein as a singlepolypeptide.

In addition, in some embodiments, the naturally occurring disulfide bondbetween Cys 76 and Cys 175 may be removed in hNGAL muteins of thedisclosure. Accordingly, such muteins can be produced in a cellcompartment having a reducing redox milieu, for example, in thecytoplasma of Gram-negative bacteria.

In case a mutein of the disclosure includes intramolecular disulfidebonds, it may be preferred to direct the nascent polypeptide to a cellcompartment having an oxidizing redox milieu using an appropriate signalsequence. Such an oxidizing environment may be provided by the periplasmof Gram-negative bacteria such as E. coli, in the extracellular milieuof Gram-positive bacteria or in the lumen of the endoplasmatic reticulumof eukaryotic cells and usually favors the formation of structuraldisulfide bonds.

It is, however, also possible to produce a mutein or polypeptide thereofof the disclosure in the cytosol of a host cell, preferably E. coli. Inthis case, the mutein or polypeptide can either be directly obtained ina soluble and folded state or recovered in form of inclusion bodies,followed by renaturation in vitro. A further option is the use ofspecific host strains having an oxidizing intracellular milieu, whichmay thus allow the formation of disulfide bonds in the cytosol (Venturiet al. (2002) J. Mol. Biol. 315, 1-8.).

However, the mutein or polypeptide as described herein may notnecessarily be generated or produced only by use of genetic engineering.Rather, such mutein or polypeptide can also be obtained by chemicalsynthesis such as Merrifield solid phase polypeptide synthesis or by invitro transcription and translation. It is for example possible thatpromising mutations are identified using molecular modeling and then tosynthesize the wanted (designed) polypeptide in vitro and investigatethe binding activity for pyoverdine type I, II, III or pyochelin.Methods for the solid phase and/or solution phase synthesis of proteinsare well known in the art (see e.g. Bruckdorfer, T. et al. (2004) Curr.Pharm. Biotechnol. 5, 29-43).

In another embodiment, the mutein or polypeptide of the disclosure maybe produced by in vitro transcription/translation employingwell-established methods known to those skilled in the art.

The skilled worker will appreciate methods useful to prepare muteins orpolypeptides thereof contemplated by the present disclosure but whoseprotein or nucleic acid sequences are not explicity disclosed herein. Asan overview, such modifications of the amino acid sequence include,e.g., directed mutagenesis of single amino acid positions in order tosimplify sub-cloning of a mutated hNGAL gene or its parts byincorporating cleavage sites for certain restriction enzymes. Inaddition, these mutations can also be incorporated to further improvethe affinity of a mutein for its target (e.g. pyoverdine or pyochelin,respectively). Furthermore, mutations can be introduced to modulatecertain characteristics of the mutein such as to improve foldingstability, serum stability, protein resistance or water solubility or toreduce aggregation tendency, if necessary. For example, naturallyoccurring cysteine residues may be mutated to other amino acids toprevent disulphide bridge formation.

The muteins or polypeptides thereof disclosed herein and theirderivatives can be used in many fields similar to antibodies orfragments thereof. For example, the muteins can be used for labelingwith an enzyme, an antibody, a radioactive substance or any other grouphaving biochemical activity or defined binding characteristics. By doingso, their respective targets or conjugates or fusion proteins thereofcan be detected or brought in contact with them. In addition, muteins orpolypeptides thereof of the disclosure can serve to detect chemicalstructures by means of established analytical methods (e.g., ELISA orWestern Blot) or by microscopy or immunosensorics. In this regard, thedetection signal can either be generated directly by use of a suitablemutein conjugate or fusion protein or indirectly by immunochemicaldetection of the bound mutein via an antibody.

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing Examples and the attached Figures thereof, which are notintended to be limiting. Thus, it should be understood that although thepresent disclosure is specifically disclosed by exemplary embodimentsand optional features, modification and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure.

V. EXAMPLES Example 1: Purification and Biotinylation of Pseudomonasaeruginosa Siderophores

P. aeruginosa produces three groups of pyoverdines i.e. pyoverdine typeI, pyoverdine type II & pyoverdine type III. Each group has three formsdiffering in the side chain which is succinyl, succinamid orα-ketoglutaryl. In addition P. aeruginosa produces pyochelin. All tensiderophores can complex iron as Fe³.

For selection and screening of muteins of interest, the siderophores maybe biotinylated. Biotinylation was performed for pyoverdine I succinylvariant at the succinyl side chain, for pyoverdine II succinyl variantat the L-ornithine side chain and for pyoverdine III succinyl variantmainly at the glycine side chain. Pyochelin was biotinylated at thephenol ring.

Example 2: Selection of Muteins Specifically Binding to P. aeruginosaSiderophores

hNGAL-based libraries, generated by random mutagenesis of mature hNGAL,were used for selection of muteins specifically binding to the differentsiderophores of P. aeruginosa. Biotinylated and iron loaded Pvd Isuccinyl. Pvd II succinyl, and Pvd III succinyl as well as biotinylatednon-iron-loaded pyochelin were used in independent phage display andselection processes.

2×10¹² phagemids from these libraries were incubated with 200 nM or 500nM or 1 μM biotinylated target. Paramagnetic beads coated withneutravidin or streptavidin were used to capture target/phagemidcomplexes which were subsequently isolated with a magnet. Unboundphagemids were removed by washing the beads with PBST or PBS. Boundphagemids were first eluted with 300 μl 70 mM triethylamine for 10 minfollowed by immediate neutralization of the supernatant with 100 μl 1MTris-Cl pH 6.0. After one intermediate wash cycle remaining phagemidswere eluted with 100 mM glycin pH2.2 for 10 min followed by immediateneutralization with 50 μl 0.5 M Tris-base. Both elution fractions werepooled and used to infect 4 ml of E. coli XL1-blue culture (OD₅₅₀0.45-0.6) for reamplification. After incubation for 30 min underagitation bacteria were collected by centrifugation at 5000×g for 2 min,resuspended in 1 ml 2xYT medium and plated on three big LB/Amp agarplates (10 g/l bacto tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.5,15 g/l agar, 100 μg/ml ampicillin). Plates were incubated overnight at32° C. Infected cells were scraped from the agar plates using 50 ml 2xYTmedium supplemented with 100 μg/ml ampicillin (2xYT/Amp). 50 ml 2xYT/Ampmedium were inoculated with the appropriate volume of bacterialsuspension to reach an OD₅₅₀ of 0.08. The culture was incubated at 37°C. on a shaker (160 rpm) until an OD₅₅₀ of 0.5 was reached and theninfected with helperphages (1.5×10¹¹ pfu) by incubation for 15 min withgentle agitation and for 45 min on a shaker at 37° C. Subsequently,kanamycin was added to a final concentration of 70 μg/ml to selectbacteria infected by helperphages. Finally, expression of the pIII-hNGALmuteins was induced by addition of 25 ng/ml anhydrotetracyclin.

After 15 h incubation at 24° C. the supernatant of the culture wascleared by centrifugation (5000×g for 20 min). Subsequently, 20 mlsupernatant were passed through a polyethersulfone membrane with a poresize of 0.22 μm. To the filtrate 5 ml of a solution containing 20% (w/v)PEG-8000 and 15% (w/v) NaCl in water was added and gently mixed. Thesolution was incubated for 30 min on ice before centrifugation for 20min at 4° C. & 5000×g. The pellet containing the phagemids was dissolvedin 1 ml buffer containing 200 mM boric acid, 160 mM NaCl and 1 mM EDTA.Unsoluble particles were removed by centrifugation (5000×g for 5 min).The supernatant was transferred to a fresh tube and mixed with 200 μl ofa solution containing 20% (w/v) PEG-8000 and 15% (w/v) NaCl in water.The solution was incubated 30 min on ice and precipitated phagemids weresubsequently collected by centrifugation (5000×g for 5 min). Phagemidswere resuspended in PBS supplemented with 50 mM benzamidine and used forthe next round of phagemid selection.

Four consecutive rounds of selection were performed. Different washingconditions were applied: i) eight times with 1 ml PBS/T 5 min incubationfor each washing step in all 4 selection rounds, ii) the number of washcycles increased from round 1 to 4 iii) fast washing steps were alteredwith 5 min incubation washing steps and the number of washings steps wasincreased from round to round.

Phagemid DNA was prepared from E. coli cells infected with the output ofthe fourth selection round and the hNGAL mutein cassette was isolated bydigestion of the DNA with BstX1 and subsequent purification via agarosegel electrophoresis using standard methods (Sambrook et al., (1989)Molecular cloning: a laboratory manual). The hNGAL mutein cassette wasinserted into the likewise cut vector, which allows bacterial productionof the hNGAL muteins under the control of a tetracyclin promoter.CaCl₂-competent TG1-F′ cells were transformed with the ligation mixtureand plated on LB/Amp plates.

For optimization of Pvd I, Pvd II, Pvd III and Pch-specific muteins,additional libraries were generated based on mutein SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 42, SEQ ID NO: 55, SEQID NO: 56 and subsequently SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO:45. Libraries were generated using either a biased randomization ofselected positions or error prone polymerase chain reaction (PCR) basedmethods. Selection of muteins was performed as described but withincreased stringency.

In order to facilitate expression in eukaryotic cells, potentialN-glycosylation sites (Asn-X-Ser/Thr) were removed.

Furthermore, mutations were introduced to further optimize forstability.

Example 3: Identification of Muteins Specifically Binding to theRespective P. aeruginosa Siderophores Using High-Throughput ELISAScreening

Individual colonies were used to inoculate 2xYT/Amp medium and grownovernight (14-18 h) to stationary phase. Subsequently, 50 μl 2xYT/Ampwere inoculated from the stationary phase cultures and incubated for 3 hat 37° C. and then shifted to 22° C. until an OD₅₉₅ of 0.6-0.8 wasreached. Production of muteins was induced by addition of 10 μl 2xYT/Ampsupplemented with 1.2 μg/ml anhydrotetracyclin. Cultures were incubatedat 22° C. until the next day. After addition of 40 μl of 5% (w/v) BSA inPBS/T and incubation for 1 h at 25° C. cultures were ready for use inscreening assays.

Specific binding of the isolated muteins to the respective siderophoretargets was tested by coating a 1:1 mixture of neutravidin andstreptavidin (5 μg/ml in PBS) overnight at 4° C. on microtiterplates.After blocking the plate 1 h with 2% BSA in PBST the respectivebiotinylated siderophore target used for selection was captured on thecoated microtiterplates at a concentration of 1.5-2.5 μg/ml in PBS/T.Plates coated in the same manner with biotinylated-aldosterone were usedas negative control target in the screening. Subsequently, 20 μl ofBSA-blocked cultures were added to the coated microtiter platecontaining either captured target or aldosterone and incubated for 1 hat 25° C. Bound muteins were detected after I h incubation with anti-T7antibody conjugated with horseradish peroxidase (Merck KgaA, Darmstadt)or anti-Streptag antibody conjugated with horseradish peroxidase (IBA,Boettingen). For quantification, 20 μl of QuantaBlu fluorogenicperoxidase substrate was added and the fluorescence determined at anexcitation wavelength of 320 nm and an emission wavelength of 430 nm.Muteins specifically binding to the respective siderophore targets werethen sequenced.

To select for muteins with increased affinity and stability screeningwas performed with i) reduced antigen concentration and/or ii)competition with unbiotinylated target and/or iii) incubation of thescreening supernatant at 65° C. or 70° C. before addition to the targetplate and/or iv) using reverse screening formats were the muteins werecaptured via the Streptag on microtiter plates coated with anti-Streptagantibody and different concentrations of biotinylated target was addedand detected via extravidin-HRP (Sigma Aldrich, St. Louis, Mo.).

Example 4: Expression of Muteins

Unique muteins were expressed with C-terminal sequence SAWSHPQFEK (SEQID NO: 127; including the SA linker and the Strep-tag® II, WSHPQFEK (SEQID NO: 128) in E. coli in 2YT-Amp media to purify the muteins afterexpression using Streptactin affinity chromatography and preparativesize exclusion chromatography were applicable.

Example 5: Affinity of Muteins to Soluble P. aeruginosa SiderophoresDetermined in an ELISA Based Setting

Solution binding of muteins was assayed by a “Solution binding ELISA”,the principle of which was as follows: a constant concentration of thetested mutein was incubated with variable concentrations of ligands (PvdI s, sa, aKG+/−Fe/Pvd II s, sa, aKG+/−Fe/Pvd III s, sa,aKG+/−Fe/Pch+/−Fe) for 1 h. After this pre-incubation in solution, analiquot of the mutein/ligand mixture was transferred to an ELISA platewith biotinalyted Pvd I s (+Fe), Pvd II s (+Fe), Pvd III s (+Fe) or Pchimmobilized via Neutravidin to measure the remaining concentration offree muteins. The concentration of free (non ligand-bound) muteins wasdetermined via a quantitative ELISA setup.

In detail, a 384-well plate suitable for fluorescence measurements(Greiner FLUOTRAC™ 600, black flat bottom, high-binding) was coated with20 μl of Neutravidin at a concentration of 5 μg/ml in PBS over night at4 C. After washing, the Neutravidin-coated wells were blocked with 100μl blocking buffer containing 0.1% Tween 20 and 2% BSA (PBS-TIBSA) for 1h at room temperature. After washing again, 20 μl biotinylatedpyoverdine or pyochelin in blocking buffer at a concentration of 1 μg/mLwere added for 1 h at room temperature and excess reagent was removed.

A fixed concentration of muteins was incubated in solution with varyingconcentrations of ligand (Pvd I s, sa, aKG+/−Fe/Pvd II s, sa,aKG+/−Fe/Pvd III s, sa, aKG+/−Fe/Pch+/−Fe), using a suitable startingconcentration which was serially diluted at a 1:3 ratio down to thepicomolar range in PBS-T/BSA. After 1 h incubation at room temperature,20 μl of the reaction mixture was transferred to the 384-well plate uponwhich biotinylated pyoverdin or pyochelin was immobilized to captureunbound (free) muteins for 20 min at RT. To allow for transformation ofELISA readout results into absolute free mutein concentrations, astandard curve containing varying concentrations of muteins was preparedin PBS-T/BSA and incubated for 20 min on the same ELISA plate as well.

The residual supernatants were discarded and 20 μl HRP-labeledanti-hNGAL antibody was added at a predetermined optimal concentrationin PBS-T/BSA and incubated for 1 h at RT. The anti-hNGAL antibody hadbeen obtained by immunization of rabbits with a mixture of muteins, andwas subsequently coupled to HRP using a kit (EZ-link Plus ActivatedPeroxidase, Thermo Scientific) according to the manufacturer'sinstructions, to obtain the antibody-HRP conjugate. After washing, 20 μlfluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well,and the reaction was allowed to proceed for 15 to 60 minutes. Thefluorescence intensity of every well on the plate was read using afluorescence microplate reader (Tecan or Molecular Devices). To evaluatethe data, free mutein concentration, c(mutein)_(free), was calculatedbased on the standard curve results, and plotted versus ligandconcentration, c(Ligand). To obtain the ligand concentration at whichformation the ligand/mutein complex was blocked by 50% (IC50), thecurves were fitted by nonlinear regression with a single-sites bindingmodel according to c(mutein)_(free)=c(mutein)_(tot)/(1+c(Ligand)/IC50)),with the total tracer concentration c(mutein)_(tot) and the IC50 valueas free parameters. Curve fitting was performed using GraphPad Prism 4software.

The resulting IC₅₀ values are summarized in Tables 1A-D. Muteinsselected against biotinylated and iron loaded Pvd I succinyl, Pvd IIsuccinyl and Pvd III succinyl, respectively bound to all subtypes of therespective Pvd group i.e. muteins selected against biotinylated and ironloaded Pvd I succinyl bound with similar affinity to Pvd I succinyl,-succinamid, -α-ketoglutaryl with or without complexed iron ion, muteinsselected against biotinylated and iron loaded Pvd II succinyl bound withsimilar affinity to Pvd II succinyl, -succinamid, -α-ketoglutaryl withor without complexed iron ion and muteins selected against biotinylatedand iron loaded Pvd III succinyl bound with similar affinity to Pvd IIIsuccinyl, -succinamid, -α-ketoglutaryl with or without complexed ironion. Most of the selected muteins bound with comparable affinity to allsubtypes of the respective group with or without complexed iron ion.

The selection against biotinylated non-iron-loaded pyochelin resulted inlipocalin muteins binding preferably to non iron-loaded pyochelin, suchas lipocalin muteins SEQ ID NO: 56 and 57 binding with two- to threedigit nM affinity to iron-loaded Pch and with weak affinity or not atall to non-iron loaded Pch, and in lipocalin muteins such as SEQ ID NO:55 binding preferably to iron-loaded pyochelin.

Affinity optimization of SEQ ID NO: 56 resulted in lipocalin muteinsbinding with improved affinity to non-iron loaded Pch and still with noor weak affinity to iron loaded Pch, whereas affinity optimization ofSEQ ID NO: 55 resulted in lipocalin muteins binding with more than 75fold improved affinity to non-iron loaded Pch but also with single digitnM affinity to iron loaded Pch.

Thus, with lipocalin mutein selection and optimization it wasaccomplished that only four different muteins are sufficient to bind all10 subtypes of P. aeruginosa siderophores with and without complexediron ion (Pvd I s, sa, αKG+/−Fe; Pvd II s, sa, (αKG+/−Fe; Pvd III s, sa,αKG+/−Fe; Pch+/−Fe).

TABLE 1A Binding of muteins to P. aeruginosa siderophore pyoverdine Isuccinyl, - succinamid, -α-ketoglutaryl +/−Fe³⁺ in solution Solutionbinding ELISA IC50: nM Pvd I Pvd I Pvd I Pvd I Pvd I s sa aKG Pvd I s saaKG (+Fe) (+Fe) (+Fe) (−Fe) (−Fe) (−Fe) SEQ ID NO: 2 24 19 13 26 19 13SEQ ID NO: 4 97 43 91 57 28 50 SEQ ID NO: 5 97 49 73 57 32 42 SEQ ID NO:6 44 30 37 48 31 36 SEQ ID NO: 7 173 126 59 290 129 53 SEQ ID NO: 8 2.381.33 2.15 2.3 0.98 1.8 SEQ ID NO: 9 3.3 1.37 2.4 3.7 1.6 2.9 SEQ ID NO:10 3.4 1.1 2.87 3.8 0.92 2.9 SEQ ID NO: 11 2.97 1.9 2.57 4 2 3.1 SEQ IDNO: 12 6.8 4.7 6.4 6.9 4.8 5.6 SEQ ID NO: 13 0.5 0.27 0.37 0.36 0.2 0.24SEQ ID NO: 14 2.4 1.7 3.1 2.4 1.1 2.2 SEQ ID NO: 15 1.1 0.59 1.2 0.860.42 0.69 SEQ ID NO: 16 1.3 0.84 1.6 1 0.63 0.83 SEQ ID NO: 18 5.3 2.23.9 2.8 1.8 2.5

TABLE 1B Binding of muteins to soluble P. aeruginosa siderophorepyoverdine II succinyl, -succinamid, -α-ketoglutaryl +/−Fe3+ in solutionSolution binding ELISA IC50: nM Pvd Pvd II Pvd II II Pvd II s sa aKG PvdII Pvd II aKG (+Fe) (+Fe) (+Fe) s (−Fe) sa (−Fe) (−Fe) SEQ ID NO: 19 3036 21 23 42 34 SEQ ID NO: 20 48 40 85 63 40 89 SEQ ID NO: 26 0.34 0.391.3 0.45 0.45 0.75 SEQ ID NO: 27 0.78 1.53 1.97 1.02 1.12 1.4 SEQ ID NO:28 0.91 1.75 2.25 1.14 1.5 1.65 SEQ ID NO: 29 0.68 1.5 1.9 0.95 1.2 1.6SEQ ID NO: 30 0.29 0.53 3 0.4 0.3 2.85 SEQ ID NO: 31 0.29 0.29 1.1 0.380.35 0.64 SEQ ID NO: 32 0.27 0.32 1.25 0.42 0.37 0.72 SEQ ID NO: 33 0.280.32 1.3 0.4 0.32 0.7 SEQ ID NO: 34 0.29 0.32 1.6 0.27 0.32 1.2 SEQ IDNO: 35 0.33 0.39 0.76 0.34 0.42 0.99 SEQ ID NO: 36 0.33 0.39 0.76 0.340.42 0.99 SEQ ID NO: 37 0.19 0.28 2.1 0.2 0.3 1.4

TABLE 1C Binding of muteins to P. aeruginosa siderophore pyoverdine IIIsuccinyl, -succinamid, -α-ketoglutaryl +/−Fe3+ in solution Solutionbinding ELISA IC50: nM Pvd Pvd Pvd III Pvd Pvd III III s III sa aKG PvdIII III sa aKG (+Fe) (+Fe) (+Fe) s (−Fe) (−Fe) (−Fe) SEQ ID NO: 39 146147 23 95 94 23 SEQ ID NO: 42 35 15 78 25 7.2 69 SEQ ID NO: 43 0.31 0.251.4 0.6 0.46 1.90 SEQ ID NO: 44 0.35 0.26 0.93 0.35 0.21 1.10 SEQ ID NO:45 0.75 0.43 1.50 0.41 0.46 1.70 SEQ ID NO: 46 0.69 0.30 1.02 0.44 0.301.20 SEQ ID NO: 47 0.37 0.30 0.82 0.17 0.28 0.58 SEQ ID NO: 48 0.28 0.220.95 0.29 0.24 0.64 SEQ ID NO: 49 0.32 0.27 0.79 0.21 0.27 0.62 SEQ IDNO: 50 0.29 0.35 0.95 0.29 0.37 0.82 SEQ ID NO: 51 0.37 0.37 0.97 0.350.34 1.1 SEQ ID NO: 52 0.32 0.31 1 0.31 0.31 1 SEQ ID NO: 53 0.21 0.250.54 0.19 0.63 0.33

TABLE 1D Binding of muteins to P. aeruginosa siderophore pyochelin +/−Fe3+ in solution Solution Binding ELISA IC50: nM pch (+Fe) pch (−Fe) SEQID NO: 55 361 N/A SEQ ID NO: 56 N/A 51 SEQ ID NO: 57 N/A 147 SEQ ID NO:58 N/A 10 SEQ ID NO: 59 N/A 11 SEQ ID NO: 60 8.6 45 SEQ ID NO: 61 5.1 42SEQ ID NO: 62 4.7 26 SEQ ID NO: 63 5.6 26

For high throughput affinity ranking, the same assay was used howeverwith less different concentrations of ligand.

Example 6: Affinity of Muteins Binding to P. aeruginosa SiderophoresDetermined in Biacore

In a Surface Plasmon Resonance (SPR) based assay a Biacore T200instrument (GE Healthcare) was used to measure the binding affinity ofmuteins to pyoverdine I succinyl, -succinamid, -α-ketoglutaryl withcomplexed iron ion or to pyoverdine II succinyl, -succinamid,-α-ketoglutaryl with complexed iron ion or to pyoverdine III succinyl,-succinamid, -α-ketoglutaryl with complexed iron ion. Muteins selectedfor binding to pyoverdines and negative control (SEQ ID NO: 64) werebiotinylated for 2 h at room temperature applying an appropriate excessof EZ-Link NHS-PEG4-Biotin (Thermo, Cat#21329) followed by separation ofnon-reacted Biotin using a Zeba Spin Desalting Plate (Thermo, Cat#21329)according to the manufactures instructions.

In the SPR affinity assay, biotinylated muteins and negative controlwere captured on a sensor chip CAP using the Biotin CAPture Kit (GEHealthcare): Sensor Chip CAP is pre-immobilized with an ssDNA oligo.Undiluted Biotin CAPture Reagent (streptavidin conjugated with thecomplementary ss-DNA oligo) was applied at a flow rate of 2 μl/min for300 s. Subsequently, 1 μg/ml to 100 μg/mL of biotinylated muteins ornegative control were applied for 300 s at a flow rate of 5 μl/min. Thereference channel was loaded with Biotin CAPture Reagent only.

To determine the binding affinity, four to five dilutions of therespective Pvd representatives (Pvd I, II, III, including succinyl,succinamid, -α-ketoglutaryl+Fe) at a concentration in the range of5-2000 nM were prepared in HBS-EP+ buffer (GE Healthcare) and applied tothe prepared chip surface. Applying a flow rate of 30 μl/min, a singlecycle or multi cycle kinetics approach was used with a sample contacttime of 120-180 s and a dissociation time of 900-2400 s. Absence ofbinding to the negative control SEQ ID NO: 64 was confirmed using a highconcentration (e.g. 1200 nM) of the respective Pvd. After ligandimmobilization, for analysis using single cycle kinetics all 4→5concentrations of Pvd were applied consecutively in ascending orderbefore the dissociation was monitored. For analysis using multi cyclekinetics 4 dilutions of Pvd were applied, each followed a dissociationphase. All measurements were performed at 25° C. Regeneration of theSensor Chip CAP surface was achieved with an injection of 6 M Gua-HClwith 0.25 M NaOH followed by an extra wash with running buffer and astabilization period of 120 s. Data were evaluated with Biacore T200Evaluation software (V 1.0). Double referencing was used. A 1:1 Bindingmodel was used to fit the raw data.

The resulting kinetic constants for a selection of lipocalin muteins aresummarized in Tables 2A-C. Lipocalin muteins could be generated for eachPvd group binding in the subnM to low single digit nM range to allsuptypes of the respective Pvd group. The natural ligand of wild typehNGAL Fe-enterobactin, however, is not bound by the Pvd specificlipocalin muteins.

TABLE 2A Kinetic constants of Pvd I specific lipocalin muteins to Pvd Isuccinyl, - succinamid, and -α-ketoglutaryl complexed with Fe³⁺. Fe- PvdI s (+Fe) Pvd I sa (+Fe) Pvd I k (+Fe) Enterobactin k_(on) k_(off) K_(D)k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) K_(D) SEQ ID [1/Ms] [1/s] [nM][1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] [nM] SEQ ID NO: 8 5.37E+04 1.79E−043.33 1.11E+05 1.20E−04 1.08 4.74E+04 2.35E−04 4.95 no bdg. SEQ ID NO: 93.31E+04 3.30E−04 9.97 8.02E+04 2.57E−04 3.20 3.80E+04 5.32E−04 14.03 nobdg. SEQ ID NO: 10 3.47E+04 4.78E−04 13.78 8.63E+04 3.04E−04 3.525.02E+04 6.31E−04 12.57 no bdg. SEQ ID NO: 11 2.84E+04 4.04E−04 14.226.76E+04 2.97E−04 4.40 3.48E+04 5.86E−04 16.84 no bdg. SEQ ID NO: 131.17E+05 6.15E−05 0.53 1.65E+05 4.24E−05 0.26 9.51E+04 8.37E−05 0.88 nobdg. SEQ ID NO: 16 3.56E+04 1.88E−04 5.28 5.43E+04 1.56E−04 2.873.14E+04 2.54E−04 8.10 no bdg.

TABLE 2B Kinetic constants of Pvd II specific lipocalin muteins to PvdII succinyl, - succinamid, and -α-ketoglutaryl complexed with Fe³⁺. Fe-Pvd II s (+Fe) Pvd II sa (+Fe) Pvd II k (+Fe) Enterobactin k_(on)k_(off) K_(D) k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) K_(D) SEQ ID[1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] [nM] SEQ ID NO: 321.15E+06 1.09E−03 0.94 1.37E+06 9.55E−04 0.7 1.09E+05 3.74E−04 3.44 nobdg. SEQ ID NO: 33 1.23E+06 1.25E−03 1.02 1.41E+06 1.04E−03 0.749.93E+04 4.16E−04 4.19 no bdg. SEQ ID NO: 35 1.31E+05 4.59E−05 0.352.48E+05 4.58E−05 0.18 4.35E+04 1.49E−04 3.42 no bdg. SEQ ID NO: 361.10E+05 4.30E−05 0.39 1.38E+05 3.67E−05 0.27 2.86E+04 5.62E−05 1.97 nobdg.

TABLE 2C Kinetic constants of Pvd III specific lipocalin muteins to PvdIII succinyl, - succinamid, and -α-ketoglutaryl complexed with Fe³⁺. Fe-Pvd III s (+Fe) Pvd III sa (+Fe) Pvd III k (+Fe) Enterobactin k_(on)k_(off) K_(D) k_(on) k_(off) K_(D) k_(on) k_(off) K_(D) K_(D) SEQ ID[1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] [1/Ms] [1/s] [nM] [nM] SEQ ID NO: 437.05E+04 1.58E−04 2.24 3.52E+04 1.07E−04 3.04 5.73E+04 3.03E−04 5.29n.d. SEQ ID NO: 44 5.62E+04 1.42E−04 2.53 3.03E+04 8.90E−05 2.944.82E+04 2.71E−04 5.64 n.d. SEQ ID NO: 45 5.90E+04 1.59E−04 2.703.27E+04 9.91E−05 3.03 4.73E+04 3.30E−04 6.99 n.d. SEQ ID NO: 468.32E+04 1.66E−04 2.00 4.36E+04 6.90E−05 1.58 7.67E+04 2.41E−04 3.15n.d. SEQ ID NO: 47 7.89E+04 7.91E−05 1.00 1.28E+05 2.52E−05 0.202.92E+04 2.62E−04 8.97 n.d. SEQ ID NO: 48 6.70E+04 1.06E−04 1.581.48E+05 9.51E−05 0.64 2.72E+04 1.58E−04 5.81 n.d. SEQ ID NO: 496.88E+04 1.05E−04 1.52 1.34E+05 1.12E−04 0.84 2.81E+04 4.29E−05 1.53n.d. SEQ ID NO: 53 5.10E+04 4.19E−05 0.82 6.73E+04 3.90E−05 0.583.88E+04 1.40E−04 3.60 no bdg.

In addition, absence of binding to various siderophores not belonging tothe respective pyoverdine subgroup (I, II. III) and to MMP-9 wasconfirmed using the assay described above by applying highconcentrations (≥1 μM) of the following analytes to the immobilizedmutein: Fe-enterobactin, desferoxamine, pyochelin, pyoverdines from therespective other subgroups, MMP-9 proform and activated MMP-9. Anoverview of this analysis is provided in Table 3.

For determination of kinetic constants and resulting K_(D) for theinteraction of mutein SEQ ID NO: 62 with Pch+Fe the mutein or thenegative control SEQ ID NO: 64 was immobilized to the surface of a CM5chip using standard amine chemistry: The surface of the chip wasactivated using EDC and NHS. Subsequently, 5 μg/mL of mutein or thenegative control solution in 10 mM acetate pH 4.0 was applied at a flowrate of 10 μl/min until a high immobilization level of approximately2000 RU was achieved. Residual activated groups were quenched withethanolamine. The reference channels were treated with EDC/NHS followingethanolamine (blank immobilization).

To determine the affinity, five dilutions of pyochelin (+Fe), wereprepared in HBS-P+ buffer and applied to the prepared chip surface. Thebinding assay was carried out with a contact time of 180 s, dissociationtimes of 1200-1800 s and applying a flow rate of 30 μl/min. Measurementswere performed at 25° C. Regeneration of the immobilized mutein surfacewas achieved by three consecutive injections of 10 mM Gly-HCl pH 1.5(120 s) followed by an extra wash with running buffer and astabilization period. Data were evaluated with Biacore T200 Evaluationsoftware (V 1.0). Double referencing was used. The 1:1 Binding model wasused to fit the raw data.

The resulting kinetic constant for SEQ ID NO: 62 is shown in Table 2D.

Using the same assay, absence of binding to siderophores different frompyochelin and to MMP-9 was confirmed by applying high concentrations (≥1μM) of the following analytes to the immobilized mutein SEQ ID NO: 62:Fe-enterobactin, desferoxamine, pyoverdine, MMP-9 proform and activatedMMP-9. An overview of the results is shown in Table 3.

TABLE 2D Kinetic constants of pyochelin specific lipocalin mutein SEQ IDNO: 62 to pyochelin complexed with Fe³⁺. pch (+Fe) k_(on) k_(off) K_(D)SEQ ID [1/Ms] [1/s] [nM] SEQ ID NO: 62 2.25E+06 6.43E−04 0.29

TABLE 3 Specificity of lipocalin muteins binding to Pvd I, Pvd II, PvdIII or pyochelin. Pvd I s Pvd II s Pvd III pch Enterobactin Desferox-proform activated (+Fe) (+Fe) s (+Fe) (+Fe) (+Fe) amin MMP-9 MMP-9 SEQ +− − − − − − − ID NO: 16 SEQ − + − − − − − − ID NO: 36 SEQ − − + − − − −− ID NO: 53 SEQ − − − + − − − − ID NO: 62 SEQ − − − − + − − − ID NO: 64

Example 7: Functional Testing of Muteins Binding to P. aeruginosaSiderophores; Inhibition of Iron Uptake

To determine the functional iron uptake inhibition in living bacteria, adose range concentration of the lipocalin muteins binding to P.aeruginosa siderophore are incubated for 1 hour with 100 nM radioactiveiron loaded siderophore in a Tris.HCl 50 mM pH 8.0 buffer before beingincubated for 30 minutes with bacteria at a final concentration of OD=1at 595 nm in a 96 well plate. Subsequently bacteria are filtered with acell harvester through a 96 well plate GF/B filter preincubated with aPoly Ethylene Imine solution at 5% and washed 3 times with Tris buffer.After filtering and drying, 30 μl of scintillant cocktail are added ineach filter well before counting. To iron load pyoverdine, siderophoreis incubated for 15 minutes with 55Fe—Cl3 in Tris buffer with a 4 to 1ratio of pyoverdine and iron in a 200 μM final solution. For loadingpyochelin with radioactive iron, a 40 μl solution of 55FeCl3 at 0.25 mMin HCl 0.5 N is added to a methanol solution of pyochelin at 1 mM. Aftera 15 minutes incubation time, 940 μl Tris HCl 50 mM pH 8.0 is added toobtain a 20 μM 55Fe-Pch solution with a 2 to 1 ratio between pyochelinand iron. The bacteria are prepared as follow: 10 ml of an overnightculture in Mueller Hinton Medium inoculated with an isolated clone iscentrifuged and the washed pellet is resuspended in 25 ml of succinatemedium and incubated under shaking for 2 hours. In parallel, 20 ml ofMueller Hinton Medium are inoculated with 5 ml of the overnight cultureand incubated under shaking for 2 hours to be used as background ironuptake level. The 25 ml bacteria cultures are then centrifuged andwashed with the corresponding medium before the pellet is resuspended inTris.HCL 50 mM pH8.0 buffer and the OD at 595 nm measured to have afinal concentration in the assay of OD=1.

Percentage of incorporation is calculated for each concentration pointand the inhibition is calculated with in-house software. For thiscalculation, the maximum level of iron uptake is based on the valueobtained in Minimum Succinate Medium without any lipocalin mutein, andthe background value is obtained in the rich Mueller Hinton Medium wherethe siderophore receptor is not expressed.

TABLE 4 Lipocalin muteins block iron uptake of P. aeruginosa asexemplarily shown for lipocalin muteins SEQ ID NO: 16, 37, 53 and 62.SEQ ID Iron uptake IC50: nM SEQ ID NO: 16 Pvd I s Pvd I sa Pvd I aKG 121123 183 SEQ ID NO: 37 Pvd II s Pvd II sa Pvd II aKG 118 107  51 SEQ IDNO: 53 Pvd III s Pvd III sa Pvd III aKG  74  32  8 SEQ ID NO: 62 Pch 54

Example 8: Functional Testing of Muteins Binding to P. aeruginosaSiderophores; Growth Inhibition

Bacterial growth inhibition is determined by incubating the muteinsbinding to P. aeruginosa siderophores in the Chelex treated SuccinateMedium complemented with a Trace Element Solution and 0.1 mg/ml BSA witha MS bacterial culture diluted at a final OD of 0.05 at 595 nm in ablack 96 well plate with transparent bottom. The plate is incubated overnight at 37° C. with an every 20 minutes shaking and OD reading at 595nm in an IEMS Reader MF from Thermo Labsystem. Growth inhibition isexemplarily shown for a Pvd I strain and Pvd I specific mutein SEQ IDNO: 16 in FIG. 4A, for a Pvd II strain and Pvd II specific mutein SEQ IDNO: 19, and SEQ ID NO: 36 in FIG. 4B, for a Pvd III strain and Pvd IIIspecific mutein SEQ ID NO: 53 in FIG. 4C and for a Pvd I knock-out(ΔpvdA) strain relying on pyochelin for iron uptake to grow andpyochelin specific mutein SEQ ID NO: 62 in FIG. 4D. Control is bacterialgrowth without lipocalin mutein.

Example 9: Stability Assessment of Muteins

To determine melting temperatures as a general indicator for overallstability, siderophore-specific muteins (SEQ ID NOs: 13-18; 26, 31-36;47-53; 58-62) at a protein concentration of 1 mg/ml in PBS (Gibco) werescanned (25-100° C.) at 1° C./min using a capillary nanoDSC instrument(CSC 6300, TA Instruments). The melting temperature (Tm) was calculatedfrom the displayed thermogram using the integrated Nano Analyzesoftware.

The resulting melting temperatures as well as the onset of melting forthe lipcalin muteins (SEQ ID NOs: 13-18; 26, 31-36; 47-53; 58-62) arelisted in Tables 5A-D below. For all Pvd groups as well as for pchlipocalin muteins with Tms in the range of 70° C., best lipocalin muteinfor each Pvd type and pch ranging from 68 to 74° C., could be selectedindicating good stability of the molecules.

TABLE 5A Tm and onset of melting as determined by nanoDSC of Pvd Ispecific lipocalin muteins nanoDSC SEQ ID Tm ° C. onset SEQ ID NO: 13 5951 SEQ ID NO: 14 61 51 SEQ ID NO: 15 68 59 SEQ ID NO: 16 69 60 SEQ IDNO: 17 61 53 SEQ ID NO: 18 61 54

TABLE 5B Tm and onset of melting as determined by nanoDSC of Pvd IIspecific lipocalin muteins nanoDSC SEQ ID Tm ° C. onset SEQ ID NO: 26 6558 SEQ ID NO: 31 67 60 SEQ ID NO: 32 64 56 SEQ ID NO: 33 67 61 SEQ IDNO: 34 67 56 SEQ ID NO: 35 71 63 SEQ ID NO: 36 70 61

TABLE 5C Tm and onset of melting as determined by nanoDSC of Pvd IIIspecific lipocalin muteins nanoDSC SEQ ID Tm ° C. onset SEQ ID NO: 47 6253 SEQ ID NO: 48 64 55 SEQ ID NO: 49 59 50 SEQ ID NO: 50 61 52 SEQ IDNO: 51 62 53 SEQ ID NO: 52 59 49 SEQ ID NO: 53 68 59

TABLE 5D Tm and onset of melting as determined by nanoDSC of pchspecific lipocalin muteins nanoDSC SEQ ID Tm ° C. onset SEQ ID NO: 58 6351 SEQ ID NO: 59 60 54 SEQ ID NO: 60 68 56 SEQ ID NO: 61 69 63 SEQ IDNO: 62 74 63

To assess storage and freeze/thaw stability muteins at a conc. of Img/ml in PBS were incubated for 1 week at 37° C. or underwent threefreeze/thaw cycles. Active mutein was measured in a quantitative ELISAsetting. Monomeric protein was measured in an analytical size exclusionchromatography. Exemplary data for SEQ ID NO: 16, 36, 53, 62 are shownin Table 6.

For assaying protein activity the following ELISA was applied: A384-well plate suitable for fluorescence measurements (Greiner FLUOTRAC™600, black flat bottom, high-binding) was coated with 20 μL ofNeutravidin (Thermo Scientific) at a concentration of 5 μg/ml in PBSovernight at 4° C. After washing, the Neutravidin-coated wells wereblocked with 100 μl blocking buffer (2% w/v BSA in PBS containing 0.1%v/v Tween-20) for 1 h. After washing again, 20 μl of biotinylated andiron loaded pyoverdin I succinyl, pyoverdin II succinyl, pyoverdin IIIsuccinyl or biotinylated pyocheline at a concentration of 1 μg/ml inblocking buffer were added. The plate was washed and 20 μl ofappropriately diluted protein standard, unstressed reference sample orstressed sample was transferred to the ELISA plate and incubated. Toquantitate plate-bound protein, the ELISA plate was washed, residualsupernatants were discarded and 20 μl HRP-labeled anti-hNGAL antibodywas added at a predetermined optimal concentration in blocking bufferand incubated. After washing, 20 μl fluorogenic HRP substrate(QuantaBlu, Pierce) was added to each well, and the reaction was allowedto proceed for 20-30 minutes. The fluorescence intensity of every wellon the plate was read using a fluorescence microplate reader (Tecan).

Unless otherwise stated all incubation steps were performed at for 1 hat room temperature and after each incubation step the plate was washedwith 100 μl PBS-T buffer (PBS, 0.05% Tween 20) for five times using aBiotek ELx405 select CW washer.

For the ELISA described above, a calibration curve including 11dilutions typically ranging from 0.008-500 ng/mL was prepared and threedifferent, independent dilutions within the linear range of thecalibration curve were prepared for each sample. Blocking bufferoptionally supplemented with 1% human or murine plasma was used for thedilutions.

The calibration curve was fit using a 4 Parameter Logistic (4PL)nonlinear regression model and used to calculate active proteinconcentrations for the tested samples. The determined active proteinconcentrations were referenced against an unstressed sample stored atthe same concentration and in the same matrix.

Analytical size exclusion chromatography was performed on an AgilentHPLC system with two Superdex 75 5/150 GL columns (GE Healthcare) in arow using PBS (Gibco) as an eluent at a flow rate of 0.3 mL/min.

To assess storage stability in plasma muteins at a conc. of 0.5 mg/mlwere incubated for 1 week at 37° C. in human, mouse and rat plasma.Active mutein was measured in a quantitative ELISA setting as described.

All tested lipocalin muteins proved to be stable under all testedconditions.

TABLE 6 Stability after 3 freeze/thaw cycles (F/T); 1 week storage inPBS at 37° C. and 1 week storage in human (hu), mouse (mu) or rat plasmaassessed by recovery of activity in qELISA and monomer content inanalytical SEC: stable in qELISA = 100 +/− 15%; stable in aSEC = 100 +/−5% (recovery of monomer peak area compared to non-stressed referencesample); for all samples including references a monomer content of 100area percent has been detected. 3xF/T, −20° C. 1 mg/ml 1 week PBS, 37°C., 1 mg/ml % recovery % 1 week hu 1 week mu 1 week rat of activity %monomer in % recovery of monomer plasma, 37° C. plasma, 37° C. plasma,37° C. Mutein siderophore in qELISA aSEC activity in qELISA in aSEC %recovery of activity in qELISA SEQ ID NO: 16 Pvd I 102 98 86 98 86 100100 SEQ ID NO: 36 Pvd II 99 101 104 98 93 91 110 SEQ ID NO: 53 Pvd III98 99 107 102 92 83 101 SEQ ID NO: 62 pch 107 100 95 104 97 102 95

Example 10: In Vivo Potency of Lipocalin Muteins in Mouse Model

The prophylactic effect of SEQ ID NO: 19 following intravenous (i.v.)administration in a P. aeruginosa-induced pulmonary infection in micewas studied.

SEQ ID NO: 19 was administered 1 hour before infection and at time ofinfection. Lung bacteria load was evaluated 24 h after infection.

The strain used in this study was P. aeruginosa (ATCC27853). Startingfrom P. aeruginosa stored at −80° C. in PBS/15% Glycerol, an overnightculture was conducted at 37° C. under shaking in Mueller-Hinton broth,and followed by additional subculture (100 μl overnight culture+100 mlof MHB) until end of logarithmic phase of growth. The culture was washedtwice and resuspended in phosphate-buffer saline before to be frozen at1E+09 CFU/ml. For each experiment a fresh vial was thawed and inoculumverified by viable counts.

7 to 8 weeks-old Male Swiss mice (5 animals/group) purchased fromJanvier laboratories, (Route des chênes secs. 53940 Le Genest Saint Ile,France), were allowed at least 5 days acclimatization prior to use.Animals were maintained at temperature of 22±2° C. with relativehumidity of 40-70% and 12-15 air fresh changes/hour. Light cycle 12/12hours: light 7 a.m. to 7 p.m. (normal cycle). Temperature and relativehumidity derivations are recorded continuously. Animals were housing 5per cages and they allowed access to water and standard diet (AO4 Cstandard diet (SAFE)) ad libitum. All experiments were performed withapproval of the ethic committee of Sanofi R&D (CEPAL).

Lung infection was Induced by intranasal challenge of male Swiss micewith 1.E+07 CFU/mouse of P. aeruginosa in 50 μl NaCl 0.9%.

SEQ ID NO: 19 at concentrations of 200, 400, 1000 or 2000 μg/mouse wasadministered 1 h before infection and at time of infection, with i.v.bolus.

Twenty four hours after infection, animals were euthanized and bacterialcount from lung homogenates were determined and expressed in log 10CFU/ml as mean±sem.

Statistical analysis was performed using SAS v9.2. The Excel software2003 was used for figure presentations. Comparisons on SEQ ID NO: 19doses versus vehicle were evaluated with a one-way analysis of variancefollowed by Dunnett's test (ZAR J. H., «Biostatistical Analysis»,Prentice Hall International Editions, 4ème édition, 1999.; C. W.Dunnett, “A multiple comparison procedure for comparing severaltreatments with a control”, J. Amer. Statist. Assoc., 50 (1955), pp.1096-1121, 1955).

In a P. aeruginosa-induced lung infection model in mice, SEQ ID NO: 19was administrated 1 hour before and at time of bacteria challenge andSEQ ID NO: 19 prevented the development of infection in mice in adose-dependent manner. A significant prevention effect was observed forSEQ ID NO: 19 starting at 200 μg/mouse, with a maximal effect at 2000μg/mouse.

Example 11: Crystallisation

To determine the three dimensional structure of SEQ ID NO: 31 protein incomplex with Pvd-Fe the following procedure was applied.

The protein sequence depicted in FIG. 6 was cloned in the pET-24aplasmid and expressed as N-terminally tagged 6His-TEV proteaserecognition site construct.

The plasmid was used to transform BL21(DE3) Star E. coli cells and theresulted clones were inoculated in Overnight Express Instant TB Medium(Novagen) and the cells were harvested after 47 hours of incubation at18° C. with 200 RPM agitation at final OD600 4.7. The cell pellet wasresuspended in buffer containing 500 mM NaCl, 10 mM Imidazole, 1 mMMgCl₂, 1 mM TCEP, 5% glycerol and 20 mM Tris pH 7.4 and lysed bystandard ultra-sonication procedure. The resulted extract was cleared bylow speed centrifugation and supernatant was filtered throw 22 nmmembrane before loading to Ni NTA (Qiagen) 5 ml column pre-equilibratedwith 100 mM NaCl, 10 mM Imidazole, 100 mM HEPES pH 8 buffer. The proteinwas eluted by linear gradient of imidazole 10 mM to 300 mM and furtherdialyzed overnight to 100 mM NaCl, 10 mM Imidazole, 100 mM HEPES pH 8buffer. The protein was concentrated to 20 mg/ml and loaded to Gelfiltration Superdex 75 column (GE). The resulted protein was dialyzed to100 mM NaCl, 10 mM HEPES pH 8 buffer overnight in the presence of TEVprotease (1/50 ratio) to remove 6His N-terminal tag following bynegative Ni NTA purification step as described above to separate thecleaved protein. Final protein was concentrated to 12 mg/ml in 100 mMNaCl, 50 mM HEPES pH 7.5 aliquoted, snap frozen in liquid nitrogen andstored at −80° C. for further use.

For crystallization the protein was incubated with 10× times highermolar concentration of Pvd-Fe overnight and plated for crystallizationscreening carried out in SBS format plates where 100 nL protein dropswere mixed with 100 nL of crystallization screening solution in vapordiffusion sitting drops format experiments at 20° C. and 4° C. A numberof crystallization hits were detected and crystallization conditionswere further optimized in order to obtain well diffracting x-ray qualitycrystals.

The crystals diffraction quality was assessed using synchrotron x-raysource and the best diffracting crystals were obtained under 20% PEG3350and 0.2M LiSO4 conditions at 20° C. The best crystals were cryoprotectedby increasing PEG3350 concentration to 35% than snap frozen in liquidnitrogen and 1.8 Å data set was collected at 100K temperature.

X-ray data were processed by MOSFLM and the protein structure wasdetermined by molecular replacement method using pdb 1 LKE as a searchmodel and the structural model was further refined toRfree=0.233−R=0.200 quality in P41212 with 2 ternary protein complexesper asymmetric unit.

The protein structure presents classical lipocalin scaffold with Pvd-Febound to both mutein proteins present in the asymmetric unit, FIG. 7.The amino acid residues involved in the Pvd-Fe binding analysed andpresented on FIG. 8. The oxygens of the Pvd directly binding Fe areidentified and presented on FIG. 9.

The present invention pertains to a polypeptide having bindingspecificity for pyoverdine type I, II, III or pyochelin, wherein thepolypeptide comprises an hNGAL mutein that binds pyoverdine type I. II,III or pyochelin with detectable affinity.

In one embodiment the hNGAL mutein comprises a mutated amino acidresidue at one or more positions corresponding to positions 28, 34, 36,39-42, 44-47, 49, 52, 54-55, 65, 68, 70, 72-75, 77, 79-81, 87, 96, 100,103, 106, 108, 123, 125, 127, 132, 134, 141 and 145 of the linearpolypeptide sequence of the mature hNGAL (SEQ ID NO: 1).

In another embodiment said mutein is capable of binding pyoverdine typeI complexed with iron with a K_(D) of about 20 nM or lower when measuredby Biacore T200 instrument in an assay essentially described in Example6.

In another embodiment said hNGAL mutein is capable of binding Pvd type Isuccinyl, Pvd type I succinamid and Pvd type I α-ketoglutaryl with andwithout complexed iron, with an affinity measured by an IC50 value ofabout 200 nM or lower, when measured in an ELISA assay essentiallydescribed in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting ironuptake mediated by pyoverdine type I with an IC50 value of about 150 nMor lower in a competition ELISA format essentially described in Example7.

In another embodiment the hNGAL mutein is capable of inhibitingbacterial growth of Pvd I strain in an assay essentially described inExample 8.

In another embodiment the hNGAL mutein comprises a mutated amino acidresidue at one or more positions corresponding to positions 28, 36,39-41, 46, 49, 52, 54-55, 59, 65, 68, 70, 72-75, 77, 79-81, 87, 96, 100,103, 106, 125, 127, 132, 134 and 136 of the linear polypeptide sequenceof the mature hNGAL (SEQ ID NO: 1).

In another embodiment the amino acid sequence of the hNGAL muteincomprises at least one of the following mutated amino acid residues incomparison with the linear polypeptide sequence of the mature hNGAL: Leu36→Asn, Thr, Val, Trp or Phe; Ala 40→Gly, Asn, Thr or Phe; Ile 41→Arg,Ala, Thr, Phe or Trp; Gln 49→Ile, Leu, Vla, Ala or Pro; Tyr 52→Met, Trpor Pro; Ser 68→Asp, Vla or Glu; Leu 70→Gln, Trp, Asp or Thr; Arg 72→Trp,Ala, Ser, Leu, Pro or Glu; Lys 73→Asp, Leu, Ala, Glu or Asn; Asp 77→Arg,Leu, Tyr, Ser, Gln, Thr, Ile or Asn; Trp 79→Gln, Asp, Ser, Arg, Met orGlu; Arg 81→Gln, Gly, Ile, Glu, His or Asp; Asn 96→His, Ile, Gly, Tyr orAsp; Tyr 100→Lys, Glu, Asn, Ser, Phe or Tyr; Leu 103→Lys, Pro, Gln, His,Asp, Tyr, Glu, Trp or Asn; Tyr 106→His, Gln or Phe; Lys 125→Arg, Ser,Trp, Tyr, Val or Gly; Ser 127→Trp, Asn, Ala, Thr, Tyr, His, Ile, Val orAsp; Tyr 132→Trp, Asn, Gly or Lys; and Lys 134→Asn, His, Trp, Gly, Glnor Asp.

In another embodiment the amino acid sequence of the hNGAL muteincomprises the following substitution in comparison with the linearpolypeptide sequence of the mature hNGAL: Gln 28→His; Lys 46→Glu; Thr54→Vla or Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Asp or Gln; Ile 80→Thr;Cys 87→, Ser or Asn; and Thr 136→Ala.

In another embodiment the hNGAL mutein comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutatedamino acid residues at the sequence positions 28, 36, 39-41, 46, 49, 52,54-55, 59, 65, 68, 70, 72-75, 77, 79-81, 87, 96, 100, 103, 106, 125,127, 132, 134 and 136 of the linear polypeptide sequence of the maturehuman NGAL (SEQ ID NO: 1).

In another embodiment the hNGAL mutein comprises one of the followingsets of amino acid substitutions in comparison with the linearpolypeptide sequence of the mature hNGAL:

Gln 28→His; Leu 36→Asn; Ala 40→Gly; Ile 41→Trp; Gln 49→Ile; Tyr 52→Met;Ser 68→Val; Leu 70→Gln; Arg 72→Trp; Lys 73→Asp; Asp 77→Leu; Trp 79→Gln;Arg 81→Gln; Cys 87→Ser; Asn 96→His; Tyr 100→Lys; Leu 103→His; Tyr106→His; Lys 125→Arg; Ser 127→Trp; Tyr 132→Trp; Lys 134→Asp;

Gln 28→His; Leu 36→Thr; Ala 40→Gly; Ile 41→Phe; Gln 49→Leu; Tyr 52→Trp;Leu 70→Trp; Arg 72→Ala; Lys 73→Leu; Asp 77→Tyr; Trp 79→Asp; Arg 81→Gly;Cys 87→Ser; Asn 96→Ile; Tyr 100→Glu; Leu 103→His; Tyr 106→Gln; Lys125→Trp; Ser 127→Asn; Tyr 132→Asn; Lys 134→Gln;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Ser; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Lys; Tyr106→His; Lys 125→Tyr; Ser 127→Ala; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Phe; Ala 40→Asn; Ile 41→Arg; Gln 49→Pro; Tyr 52→Met;Ser 68→Asp; Leu 70→Thr; Arg 72→Glu; Lys 73→Ala; Asp 77→Arg; Trp 79→Arg;Arg 81→Ile; Cys 87→Ser; Asn 96→Tyr; Tyr 100→Lys; Leu 103→Pro; Tyr106→Phe; Lys 125→Ser; Ser 127→Thr; Tyr 132→Trp; Lys 134→Gly;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Val; Tyr 52→Met; Ser 68→Val;Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu;Cys 87→Ser; Asn 96→Asp; Tyr 100→Phe; Leu 103→Trp; Tyr 106→Gln; Lys125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Gln 28→His; Leu 36→Val; Ala 40→Phe; Ile 41→Phe; Gln 49→Ala; Tyr 52→Pro;Ser 68→Glu; Leu 70→Trp; Arg 72→Leu; Lys 73→Asn; Asp 77→Gln; Trp 79→Glu;Arg 81→His; Cys 87→Ser; Asn 96→Tyr; Leu 103→Tyr; Tyr 106→His; Lys125→Val; Ser 127→His; Tyr 132→Lys; Lys 134→Trp;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Ser; Trp 79→Ser;Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ser; Leu103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Ile; Tyr 132→Gly; Lys134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Ser; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asp; Tyr106→His; Lys 125→Tyr; Ser 127→Val; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Asp 77→Thr; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Asp; Tyr 100→Asn; Leu 103→Glu; Tyr106→His; Lys 125→Tyr; Ser 127→Asp; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Asp; Asp 77→Val; Trp 79→Ser;Arg 81→Ile; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asn; Leu 103→Asn; Tyr106→His; Lys 125→Tyr; Ser 127→Vla; Tyr 132→Gly; Lys 134→Asn;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Gln 49→Leu; Tyr 52→Met; Ser 68→Val;Leu 70→Asp; Arg 72→Glu; Lys 73→Leu; Asp 77→Arg; Trp 79→Met; Arg 81→Glu;Cys 87→Ser; Asn 96→Asp; Tyr 100→Ser; Leu 103→Trp; Tyr 106→Gln; Lys125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Gln 28→His; Leu 36→Trp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Thr 54→Val; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Lys 75→Glu;Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly;Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Thr; Tyr132→Gly: Lys 134→Asn;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Ser 68→Val; Leu 70→Asp; Arg 72→Glu;Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met; Ile 80→Thr;Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu 103→Trp; Tyr106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys 134→His;

Leu 36→Trp; Asn 39→Asp; Ala 40→Thr; Ile 41→Thr; Gln 49→Pro; Tyr 52→Pro;Thr 54→Val; Asn 65→Asp; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu;Lys 75→Glu; Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser;Asn 96→Gly; Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser127→Thr; Tyr 132→Gly; Lys 134→Asn; Thr 136→Ala;

Leu 36→Trp; Ala 40→Thr; Ile 41→Ala; Gln 49→Pro; Tyr 52→Pro; Thr 54→Val;Asn 65→Asp; Ser 68→Asp; Leu 70→Gln; Arg 72→Ser; Lys 73→Glu; Lys 75→Glu;Asp 77→Ser; Trp 79→Ser; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Gly;Tyr 100→Ser; Leu 103→Gln; Tyr 106→His; Lys 125→Tyr; Ser 127→Thr; Tyr132→Gly; Lys 134→Asn; Thr 136→Ala;

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Asp; Ser 68→Val; Leu 70→Asp;Arg 72→Glu; Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met;Ile 80→Thr; Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys134→His; or

Gln 28→His; Ala 40→Gly; Ile 41→Trp; Lys 46→Glu; Gln 49→Leu; Tyr 52→Met;Thr 54→Ala; Ile 55→Vla; Lys 59→Arg; Asn 65→Gln; Ser 68→Val; Leu 70→Asp;Arg 72→Glu; Lys 73→Leu; Lys 74→Glu; Lys 75→Glu; Asp 77→Arg; Trp 79→Met;Ile 80→Thr; Arg 81→Glu; Ser 87→Asn; Asn 96→Asp; Tyr 100→sER; Leu103→Trp; Tyr 106→Gln; Lys 125→Gly; Ser 127→Tyr; Tyr 132→Trp; Lys134→His.

In another embodiment the hNGAL mutein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-18 or a fragment orvariant thereof.

In another embodiment said hNGAL mutein is capable of binding pyoverdinetype II complexed with iron with a K_(D) of about 20 nM or lower whenmeasured by Biacore T200 instrument in an assay essentially described inExample 6.

In another embodiment said hNGAL mutein is capable of binding Pvd typeII succinyl, Pvd type II succinamid and Pvd type II α-ketoglutaryl withand without complexed iron, with an affinity measured by an IC50 valueof about 200 nM or lower, when measured in an ELISA assay essentiallydescribed in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting ironuptake mediated by pyoverdine type II with an IC50 value of about 150 nMor lower in a competition ELISA format essentially described in Example7.

In another embodiment the hNGAL mutein is capable of inhibitingbacterial growth of Pvd II strain in an assay essentially described inExample 8.

In another embodiment said hNGAL mutein is capable of inhibiting growthof P. aeruginosa stains expressing pyoverdine type II in an assayessentially described in Example 9.

In another embodiment the hNGAL mutein comprises a mutated amino acidresidue at one or more positions corresponding to positions 28, 36,40-41, 49, 52, 54, 65, 68, 70, 72-75, 77, 79, 81, 87, 96, 100, 103, 106,125, 127, 132 and 134 of the linear polypeptide sequence of the maturehNGAL (SEQ ID NO: 1).

In another embodiment the amino acid sequence of the hNGAL muteincomprises at least one of the following mutated amino acid residues incomparison with the linear polypeptide sequence of the mature hNGAL: Leu36→Asn, Ile or Val; Ala 40→Glu, Gly, Asn, Thr or His; Ile 41→Arg, Val orThr; Gln 49→Gly, Ala or Pro; Tyr 52→Asn, Gly, Trp or Pro; Ser 68→Asp,Arg or Glu; Leu 70→Arg or Trp; Arg 72→His, Ile, Ala, Ser or Gly; Lys73→Asn, Met, Pro, Phe, Gln or Arg; Asp 77→His, Ile, Met, Lys, Gly orAsn; Trp 79→Ser, Tyr, Ala, Asp, Phe or Trp; Arg 81→Glu, Ser, Tyr or Asp;Asn 96→Met, Ile, Arg, Asp, Lys, Asn or Ala; Tyr 100→Lys, Glu, Asn, Ser,Phe or Tyr; Leu 103→Thr, Ile, Gln, Gly, Met, His, Trp or Val; Tyr106→Met, Gln, Ala, Ile, Asn, Gly, Met or Phe; Lys 125→Ala, Ile or Asn;Ser 127→Lys, Arg, Ser, Met, Asp or Asn; Tyr 132→Met, Phe, Asn, Ala, Ile,Gly or Val; and Lys 134→Trp or Tyr.

In another embodiment the amino acid sequence of the hNGAL muteincomprises the following substitution in comparison with the linearpolypeptide sequence of the mature hNGAL: Gln 28→His; Thr 54→Ala; Asn65→Asp or Gln and Cys 87→Ser.

In another embodiment the hNGAL mutein comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutatedamino acid residues at the sequence positions 28, 36, 40-41, 49, 52, 54,65, 68, 70, 72-75, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and134 of the linear polypeptide sequence of the mature human NGAL (SEQ IDNO: 1).

In another embodiment the hNGAL mutein comprises one of the followingsets of amino acid substitutions in comparison with the linearpolypeptide sequence of the natural wildtype hNGAL:

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Tyr 100→Asn; Leu 103→Gln; Tyr 106→Met; Ser127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn; Ser 68→Asp;Leu 70→Arg; Arg 72→Ile; Lys 73→Met; Asp 77→His; Trp 79→Tyr; Arg 81→Glu;Cys 87→Ser; Asn 96→Ile; Tyr 100→Asn; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Met; Lys 134→Trp;

Gln 28→His; Leu 36→Ile; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→Ala; Lys 73→Pro; Asp 77→Ile; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Met; Tyr 100→Ser; Leu 103→Gly; Tyr106→Ala; Lys 125→Lys; Tyr 132→Val; Lys 134→Trp;

Gln 28→His; Ala 40→Asn; Gln 49→Ala; Tyr 52→Pro; Ser 68→Glu; Leu 70→Arg;Arg 72→Ser; Lys 73→Gln; Asp 77→Met; Trp 79→Ala; Arg 81→Tyr; Cys 87→Ser;Asn 96→Arg; Tyr 100→Pro; Leu 103→Thr; Tyr 106→Ile; Lys 125→Lys; Ser127→Met: Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Ala 40→His; Gln 49→Ala; Tyr 52→Pro; Ser 68→Glu; Leu 70→Asp;Arg 72→Gly; Lys 73→Arg; Asp 77→His; Trp 79→Trp; Arg 81→Glu; Cys 87→Ser;Asn 96→Arg; Tyr 100→Asp; Leu 103→Met; Tyr 106→Phe; Lys 125→Ala; Ser127→Asp; Tyr 132→Asn; Lys 134→Trp;

Gln 28→His; Leu 36→Asn; Ala 40→Gly; Ile 41→Arg; Gln 49→Pro; Tyr 52→Trp;Ser 68→Arg; Leu 70→Trp; Arg 72→Asn; Lys 73→Gln; Asp 77→Lys; Trp 79→Asp;Arg 81→Glu; Cys 87→Ser; Asn 96→Asp; Tyr 100→Thr; Leu 103→Trp; Tyr106→Asn; Lys 125→Asn; Ser 127→Met; Tyr 132→Ile; Lys 134→Tyr;

Gln 28→His; Leu 36→Vla; Ala 40→Thr; Ile 41→Thr; Gln 49→Gly; Tyr 52→Gly;Ser 68→Glu; Leu 70→Arg; Arg 72→Gly; Lys 73→Arg; Asp 77→Gly; Trp 79→Trp;Arg 81→Glu; Cys 87→Ser; Asn 96→Ala; Tyr 100→Trp; Leu 103→Ile; Tyr106→Gly; Lys 125→Lys; Ser 127→Asn; Tyr 132→Val; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu 103→Val; Tyr106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Ser;Arg 81→Glu; Cys 87→Ser; Leu 103→Gln; Tyr 106→Met; Ser 127→Lys; Tyr132→Val; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Asp 77→Asn; Trp 79→Phe; Arg 81→Glu;Cys 87→Ser; Asn 96→Lys; Tyr 100→His; Leu 103→Gln; Tyr 106→Met; Ser127→Lys; Tyr 132→Ala; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Gly; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn; Trp 79→Trp;Arg 81→Glu; Cys 87→Ser; Tyr 100→Asn; Leu 103→His; Tyr 106→Met; Ser127→Lys; Tyr 132→Gly; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Phe; Asp 77→His; Trp 79→Tyr;Arg 81→Asp; Cys 87→Ser; Leu 103→Met; Tyr 106→Gln; Lys 125→Ile; Ser127→Arg; Tyr 132→Ile; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His; Trp 79→Tyr;Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys 125→Ile; Ser127→Arg; Tyr 132→Ile; Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn;Trp 79→Phe; Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu103→Val; Tyr 106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Glu; Ile 41→Val; Gln 49→Gly; Tyr 52→Pro;Asn 65→Gln; Ser 68→Glu; Leu 70→Arg; Arg 72→His; Lys 73→Asn; Asp 77→Asn;Trp 79→Phe; Arg 81→Glu; Cys 87→Ser; Asn 96→Lys; Tyr 100→Asn; Leu103→Val; Tyr 106→Met; Lys 125→Asn; Ser 127→Lys; Tyr 132→Gly; Lys134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Thr 54→Ala; Asn 65→Asp; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg;Asp 77→His; Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser: Leu 103→Thr; Tyr106→Gln; Lys 125→Ile; Ser 127→Arg; Tyr 132→Ile: Lys 134→Trp;

Gln 28→His; Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn;Thr 54→Ala; Asn 65→Gln; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg;Asp 77→His; Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr106→Gln; Lys 125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp;

Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn; Thr 54→Ala;Asn 65→Asp; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His;Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp; or

Leu 36→Val; Ala 40→Thr; Ile 41→Ile; Gln 49→Gly; Tyr 52→Asn; Thr 54→Ala;Asn 65→Gln; Ser 68→Asp; Leu 70→Arg; Arg 72→Ile; Lys 73→Arg; Asp 77→His;Trp 79→Tyr; Arg 81→Asp; Cys 87→Ser; Leu 103→Thr; Tyr 106→Gln; Lys125→Ile; Ser 127→Arg; Tyr 132→Ile; Lys 134→Trp.

In another embodiment the hNGAL mutein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-37 or a fragment orvariant thereof.

In another embodiment said mutein is capable of binding pyoverdine typeIII complexed with iron with a K_(D) of about 20 nM or lower whenmeasured by Biacore T200 instrument in an assay essentially described inExample 6.

In another embodiment said hNGAL mutein is capable of binding Pvd typeIII succinyl, Pvd type III succinamid and Pvd type III α-ketoglutarylwith and without complexed iron, with an affinity measured by an IC50value of about 200 nM or lower, when measured in an assay essentiallydescribed in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting ironuptake mediated by pyoverdine type III with an IC50 value of about 150nM or lower in a competition ELISA format essentially described inExample 7.

In another embodiment the hNGAL mutein is capable of inhibitingbacterial growth of Pvd III strain in an assay essentially described inExample 8.

In another embodiment the hNGAL mutein comprises a mutated amino acidresidue at one or more positions corresponding to positions 28, 36,40-42, 45-47, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103,105-106, 125, 127, 132, 134 and 145 of the linear polypeptide sequenceof the mature hNGAL (SEQ ID NO: 1).

In another embodiment the amino acid sequence of the hNGAL muteincomprises at least one of the following mutated amino acid residues incomparison with the linear polypeptide sequence of the mature hNGAL: Leu36→Phe or Glu; Ala 40→Trp, Leu or Arg; Ile 41→Met, Arg, Ala, Leu or Trp;Gln 49→His, Ile, Arg, Lys, Met or Pro; Tyr 52→Asn, Tyr, Arg, Ser or Met;Ser 68→Asp, Asn, Glu or Gln; Leu 70→Lys, Asn or Arg; Arg 72→Leu, Arg,Gln or Tyr; Lys 73→His, Leu, Ala, Pro, Gln or Tyr; Asp 77→Ala, Ile, Lys,Gln or Arg; Trp 79→Ser or Asp; Arg 81→His, Ala, Ser or Val; Asn 96→Met,Ile, Arg, Gly, Leu or Val; Tyr 100→Ala, Ile, Asn, Pro or Asp; Leu103→Gln, Gly, Phe or Pro; Tyr 106→Glu; Lys 125→Trp or Thr; Ser 127→Val,His, Ile, Phe or Ala; Tyr 132→Phe; and Lys 134→Trp, Gln or Glu.

In another embodiment the amino acid sequence of the hNGAL muteincomprises the following substitution in comparison with the linearpolypeptide sequence of the mature hNGAL: Gln 28→His; Leu 42→Arg; Asp45→Gly; Lys 46→Arg; Asp 47→Asn; Asn 65→Asp; Cys 87→Ser; Ser 105→Pro andThr 145→Pro.

In another embodiment the hNGAL mutein comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutatedamino acid residues at the sequence positions 28, 36, 40-42, 45-47, 49,52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 105-106, 125, 127,132, 134 and 145 of the linear polypeptide sequence of the mature humanNGAL (SEQ ID NO: 1)

In another embodiment the hNGAL mutein comprises one of the followingsets of amino acid substitutions in comparison with the linearpolypeptide sequence of the mature hNGAL:

Gln 28→His; Leu 36→Phe; Ala 40→Trp; Ile 41→Met; Gln 49→His; Tyr 52→Asn;Ser 68→Glu; Leu 70→Lys; Arg 72→Gln; Lys 73→Ala; Asp 77→Ile; Trp 79→Ser;Arg 81→His; Cys 87→Ser; Asn 96→Ile; Tyr 100→Asn; Leu 103→Gly; Tyr106→Glu; Lys 125→Trp; Ser 127→His; Tyr 132→Phe; Lys 134→Gln;

Gln 28→His; Leu 36→Phe; Ala 40→Arg; Ile 41→Trp; Gln 49→Ile; Tyr 52→Tyr;Ser 68→Gln; Leu 70→Asn; Arg 72→Trp; Lys 73→Leu; Asp 77→Ala; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Arg; Tyr 100→Ile; Leu 103→Pro; Tyr106→Glu; Lys 125→Thr; Ser 127→Ile; Tyr 132→Phe; Lys 134→Glu;

Gln 28→His; Leu 36→Phe; Ala 40→Leu; Ile 41→Leu; Gln 49→Arg; Tyr 52→Arg;Ser 68→Asp; Leu 70→Arg; Arg 72→Leu; Lys 73→Tyr; Asp 77→Ile; Trp 79→Ser;Arg 81→Ala; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ala; Leu 103→Phe; Tyr106→Glu; Lys 125→Trp; Ser 127→Ala; Lys 134→Glu;

Gln 28→His; Leu 36→Phe; Ala 40→Trp; Ile 41→Arg; Gln 49→Pro; Tyr 52→Ser;Ser 68→Asn; Leu 70→Arg; Arg 72→Trp; Lys 73→Pro; Asp 77→Arg; Trp 79→Ser;Arg 81→Ser; Cys 87→Ser; Asn 96→Met; Tyr 100→Pro; Leu 103→Gly; Tyr106→Glu; Lys 125→Trp; Ser 127→Phe; Tyr 132→Phe; Lys 134→Glu;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Lys; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Gln; Trp 79→Asp; Arg 81→Ala;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Gln; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Thr; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Arg; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Vla; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Met; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Gln 49→Lys; Tyr 52→Met;Ser 68→Glu; Leu 70→Arg; Lys 73→Tyr; Asp 77→Gln; Trp 79→Asp; Arg 81→Vla;Cys 87→Ser; Asn 96→-; Tyr 100→Glu; Leu 103→Gln; Tyr 106→Glu; Ser127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met;Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 47→Asn; Gln 49→Met;Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp; Thr145→Pro;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 45→Gly; Lys 46→Arg;Gln 49→Met; Tyr 52→Met; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77-Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met;Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 47→Asn; Gln 49→Met;Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys;Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp; Thr 145→Pro;

Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Asp 45→Gly; Lys 46→Arg;Gln 49→Met; Tyr 52→Met; Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His;Asp 77→Lys; Trp 79→Asp; Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp;Leu 103→Gln; Ser 105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys134→Trp; or

Leu 36→Glu; Ala 40→Leu; Ile 41→Ala; Leu 42→Arg; Gln 49→Met; Tyr 52→Met;Asn 65→Asp; Ser 68→Glu; Leu 70→Arg; Lys 73→His; Asp 77→Lys; Trp 79→Asp;Arg 81→Vla; Cys 87→Ser; Asn 96→Leu; Tyr 100→Asp; Leu 103→Gln; Ser105→Pro; Tyr 106→Glu; Ser 127→Val; Tyr 132→Phe; Lys 134→Trp.

In another embodiment the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 38-53 or a fragment orvariant thereof.

In another embodiment said hNGAL mutein is capable of binding pyochelincomplexed with iron with a K_(D) of about 20 nM or lower when measuredby Biacore T200 instrument in an assay essentially described in Example6.

In another embodiment said hNGAL mutein is capable of binding pyochelinwith complexed iron, with an affinity measured by an IC50 value of about500 nM or lower, when measured in an assay essentially described inExample 5.

In another embodiment said hNGAL mutein is capable of binding pyochelinwithout complexed iron, with an affinity measured by an IC50 value ofabout 200 nM or lower, when measured in an assay essentially describedin Example 5.

In another embodiment said hNGAL mutein is capable of binding pyochelinwith and without complexed iron, with an affinity measured by an IC50value of about 200 nM or lower, when measured in an assay essentiallydescribed in Example 5.

In another embodiment the hNGAL mutein is capable of inhibiting ironuptake mediated by pyochelin with an IC50 value of about 150 nM or lowerin a competition ELISA format essentially described in Example 7.

In another embodiment the hNGAL mutein is capable of inhibitingbacterial growth of Pvd I knock-out (ΔpvdA) in an assay essentiallydescribed in Example 8 In another embodiment the hNGAL mutein comprisesa mutated amino acid residue at one or more positions corresponding topositions 28, 34, 36, 40-41, 44-46, 49, 52, 54, 65, 68, 70, 72-74, 77,79-81, 87, 96, 100, 103, 106, 108, 123, 125, 127, 132, 134 and 141 ofthe linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 1).

In another embodiment the amino acid sequence of the hNGAL muteincomprises at least one of the following mutated amino acid residues incomparison with the linear polypeptide sequence of the mature hNGAL: Leu36→His, Met or Val; Ala 40→Ile, Gln, Tyr or Phe; Ile 41→Leu, His or Trp;Gln 49→His, Arg, Ser or Ala; Tyr 52→Leu, Trp or Pro; Ser 68→Asp or His;Leu 70→Arg or Trp; Arg 72→His, Ile, Ala, Ser or Gly; Lys 73→Asn, Met,Pro, Phe, Gln or Arg; Asp 77→Arg, Thr, Pro or Asp; Trp 79→Ala, Arg, Lysor Asp; Arg 81→Thr, Ile or Trp; Asn 96→Met, Asn, Pro or Ala; Tyr100→Gly, His or Glu; Leu 103→Gly, Met, His or Gln: Tyr 106→Met. Gly, Argor Trp; Lys 125→Trp, Phe, Gly or Leu; Ser 127→Arg, Trp, Asp or Ile; Tyr132→Ala, Glu or Thr; and Lys 134→Leu, Val, Asn or Phe.

In another embodiment the amino acid sequence of the hNGAL muteincomprises the following substitution in comparison with the linearpolypeptide sequence of the mature hNGAL: Gln 28→His; Val 34→Leu; Glu44→Gly; Asp 45→Gly; Lys→Arg or Tyr; Asn 65→Asp; Ile 80→Thr; Cys 87→Ser;Leu 94→Phe; Val 108→Ala; Phe 123 Ser and Thr 141→Ala.

In another embodiment the hNGAL mutein comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 mutatedamino acid residues at the sequence positions 28, 34, 36, 40-41, 44-46,49, 52, 54, 65, 68, 70, 72-74, 77, 79-81, 87, 96, 100, 103, 106, 108,123, 125, 127, 132, 134 and 141 of the linear polypeptide sequence ofthe mature human NGAL (SEQ ID NO: 1).

In another embodiment the hNGAL mutein comprises one of the followingsets of amino acid substitutions in comparison with the linearpolypeptide sequence of the mature hNGAL:

Gln 28→His; Ala 40→Ile; Ile 41→Leu; Gln 49→His; Tyr 52→Leu; Ser 68→His;Leu 70→Thr; Arg 72→Lys; Lys 73→Trp; Asp 77→Ile; Trp 79→Ser; Arg 81→His;Cys 87→Ser; Asn 96→Met; Tyr 100→Asn; Leu 103→His; Tyr 106→Met; Lys125→Trp; Ser 127→Asp; Tyr 132→Glu; Lys 134→Leu;

Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Gln 49→Arg; Tyr 52→Trp;Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile; Asp 77→His; Trp 79→Arg;Arg 81→Thr; Cys 87 . . . . Ser; Tyr 100→His; Leu 103→Gly; Tyr 106→Gly;Lys 125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys 134→Phe;

Gln 28→His; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser; Tyr 52→Pro;Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala; Trp 79→Lys;Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly; Leu 103→Met; Tyr106→Trp; Lys 125→Gly; Ser 127→Trp; Tyr 132→Thru; Lys 134→Val;

Gln 28→His; Leu 36→Val; Ala 40→Tyr; Ile 41→Trp; Gln 49→Ala; Ser 68→Asp;Leu 70→Arg; Arg 72→Trp; Lys 73→Arg; Asp 77→Arg; Trp 79→Asp; Arg 81→Trp;Cys 87→Ser; Asn 96→Pro; Tyr 100→Glu; Leu 103→Gln; Tyr 106→Arg; Lys125→Leu; Ser 127→Arg; Tyr 132→Ala; Lys 134→Asn;

Gln 28→His; Vla 34→Leu; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser;Tyr 52→Pro; Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala;Trp 79→Lys; Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly;Leu 103→Met; Tyr 106→Trp; Phe 123→Ser; Lys 125→Gly; Ser 127→Trp; Tyr132→Thru; Lys 134→Val; Thr 141→Ala;

Gln 28→His; Leu 36→Met; Ala 40→Phe; Ile 41→His; Gln 49→Ser; Tyr 52→Pro;Ser 68→His; Leu 70→Pro; Arg 72→Trp; Lys 73→Ala; Asp 77→Ala; Trp 79→Lys;Ile 80→Thr; Arg 81→Ile; Cys 87→Ser; Asn 96→Ala; Tyr 100→Gly; Leu103→Met; Tyr 106→Trp; Phe 123→Ser; Lys 125→Gly; Ser 127→Trp; Tyr132→Thru; Lys 134→Val;

Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Asp 45→Gly; Lys 46→Arg;Gln 49→Arg; Tyr 52→Trp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile;Asp 77→Leu; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Tyr 100→His; Leu103→Gly; Tyr 106→Gly; Lys 125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys134→Phe;

Gln 28→His; Leu 36→His; Ala 40→Gln; Ile 41→Trp; Glu 44→Gly; Lys 46→Tyr;Gln 49→Arg; Tyr 52→Trp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile;Lys 74→Glu; Asp 77→His; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Leu 94→Phe;Tyr 100→His; Leu 103→Gly; Tyr 106→Gly; Val 108→Ala; Lys 125→Phe; Ser127→Ile; Tyr 132→Ala; Lys 134→Phe; or

Leu 36→His; Ala 40→Gln; Ile 41→Trp; Asp 45→Gly; Lys 46→Arg; Gln 49→Arg;Tyr 52→Trp; Asn 65→Asp; Ser 68→Asp; Leu 70→Asp; Arg 72→Ala; Lys 73→Ile;Asp 77→Leu; Trp 79→Arg; Arg 81→Thr; Cys 87→Ser; Tyr 100→His; Leu103→Gly; Tyr 106→Gly; Lys 125→Phe; Ser 127→Ile; Tyr 132→Ala; Lys134→Phe.

In another embodiment the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 54-63 or a fragment orvariant thereof.

In another embodiment the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-63 or a fragment orvariant thereof.

In another embodiment said hNGAL mutein comprises one or more non-nativecysteine residues substituting one or more amino acids of a wild typehNGAL.

In another embodiment said hNGAL mutein comprises at least one aminoacid substitution of a native cysteine residue by another amino acid.

In another embodiment said another amino acid is a serine residue.

In another embodiment the hNGAL mutein is conjugated to a compoundselected from the group consisting of an organic molecule, an enzymelabel, a radioactive label, a colored label, a fluorescent label, achromogenic label, a luminescent label, a hapten, digoxigenin, biotin, acytostatic agent, a toxins, a metal complex, a metal, and colloidalgold.

In another embodiment the hNGAL mutein is fused at its N-terminus and/orits C-terminus to a fusion partner which is a protein, or a proteindomain or a peptide.

In another embodiment the hNGAL mutein is conjugated to a compound thatextends the serum half-life of the polypeptide.

In another embodiment the polypeptide comprises a compound that extendsthe serum half-life is selected from the group consisting of apolyalkylene glycol molecule, hydroethylstarch, a Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding peptide, and an albumin bindingprotein.

In another embodiment the polyalkylene glycol is polyethylene (PEG) oran activated derivative thereof.

In another embodiment a nucleic acid molecule is encompassed comprisinga nucleotide sequence encoding any of the polypeptides mentioned herein.

In another embodiment the nucleic acid molecule is operably linked to aregulatory sequence to allow expression of said nucleic acid molecule.

In another embodiment the nucleic acid molecule is comprised in a vectoror in a phagemid vector.

In another embodiment a host cell is encompassed containing a nucleicacid molecule of any one of the ones mentioned herein.

In another embodiment a method of producing any of the polypeptidedescribed herein is encompassed, wherein the polypeptide is producedstarting from the nucleic acid coding for the polypeptide by means ofgenetic engineering methods.

In another embodiment the polypeptide is produced in a bacterial oreucaryotic host organism and is isolated from this host organism or itsculture.

In another embodiment a composition is encompassed comprising one ormore polypeptides selected from the group consisting of (i) apolypeptide specific for pyoverdine type I, (ii) a polypeptide specificfor pyoverdine type II, (iii) a polypeptide specific for pyoverdine typeIII and (iv) a polypeptide specific for pyochelin.

In another embodiment the composition comprises two or more polypeptidesselected from the group consisting of (i) a polypeptide specific forpyoverdine type I, (ii) a polypeptide specific for pyoverdine type II,(iii) a polypeptide specific for pyoverdine type III and (iv) apolypeptide specific for pyochelin.

In another embodiment the composition comprises three or fourpolypeptides selected from the group consisting of (i) a polypeptidespecific for pyoverdine type I, (ii) a polypeptide specific forpyoverdine type II, (iii) a polypeptide specific for pyoverdine type IIIand (iv) a polypeptidespecific for pyochelin.

In another embodiment the composition comprises the polypeptide specificfor pyoverdine type I.

In another embodiment the composition comprises the polypeptide specificfor pyoverdine type II.

In another embodiment the composition comprises the polypeptide specificfor pyoverdine type III.

In another embodiment the composition comprises the polypeptide specificfor pyochelin.

In another embodiment said composition further includes at least onepharmaceutically acceptable adjuvant, diluent or carrier.

In another embodiment the method of binding pyoverdine type I, II, IIIand/or pyochelin in a subject comprises administering to said subject aneffective amount of any of the compositions mentioned herein.

In another embodiment a method is encompassed for inhibiting orlessening growth of P. aeruginosa in a subject, comprising administeringto said subject an effective amount of the composition of of any of theones mentioned herein.

In another embodiment a kit is encompassed comprising one or morecontainers, separately or in admixture, and the composition of any ofthe ones mentioned herein.

In another embodiment the use of (i) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type I,(ii) a polypeptide according to any polypeptide mentioned herein cabableof binding to pyoverdine type II, (iii) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type IIIand/or (iv) a polypeptide according to any polypeptide mentioned hereincabable of binding to pyochelin is encompassed, for the binding ofpyoverdine type I, II, III and/or pyochelin in a subject.

In another embodiment the use of (i) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type I,(ii) a polypeptide according to any polypeptide mentioned herein cabableof binding to pyoverdine type II, (iii) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type IIIand/or (iv) a polypeptide according to any polypeptide mentioned hereincabable of binding to pyochelin is encompassed, for preventing orreducing iron-uptake by P. aeruginosa through pyochelin and/orpyoverdine in a subject.

In another embodiment the use of (i) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type I,(ii) a polypeptide according to any polypeptide mentioned herein cabableof binding to pyoverdine type II, (iii) a polypeptide according to anypolypeptide mentioned herein cabable of binding to pyoverdine type IIIand/or (iv) a polypeptide according to any polypeptide mentioned hereincabable of binding to pyochelin is encompassed, for the treatment oralleviation of P. aeruginosa biofilm infection in a subject.

In another embodiment the P. aeruginosa biofilm infection is acute orchronic infection.

In another embodiment said first, second, third and/or fourthpolypeptides are administered in combination, including concurrently,concomitantly or in series.

In another embodiment said first, second, third and/or fourthpolypeptides are administered independent from each other, including atindividual intervals at independent points of time.

In another embodiment a combination comprising (i) a polypeptideaccording to any polypeptide mentioned herein cabable of binding topyoverdine type I, (ii) a polypeptide according to any polypeptidementioned herein cabable of binding to pyoverdine type II, (iii) apolypeptide according to any polypeptide mentioned herein cabable ofbinding to pyoverdine type III and/or (iv) a polypeptide according toany polypeptide mentioned herein cabable of binding to pyochelin.

Embodiments illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present embodiments have been specificallydisclosed by preferred embodiments and optional features, modificationand variations thereof may be resorted to by those skilled in the art,and that such modifications and variations are considered to be withinthe scope of the invention. All patents, patent applications, textbooksand peer-reviewed publications described herein are hereby incorporatedby reference in their entirety. Furthermore, where a definition or useof a term in a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso forms part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein. In addition, where features aredescribed in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Furtherembodiments will become apparent from the following claims.

1-12. (canceled)
 13. A nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide comprising a human neutrophilgelatinase-associated lipocalin (hNGAL) mutein polypeptide havingbinding specificity for pyoverdine type III, wherein the hNGAL muteincomprises 10 or more mutations at positions 36, 40, 41, 42, 49, 52, 65,68, 70, 73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQID NO:
 1. 14. A host cell containing a nucleic acid molecule of claim13.
 15. The nucleic acid molecule of claim 13, wherein the nucleic acidmolecule encodes an hNGAL mutein comprises ten or more of the followingmutations of SEQ ID NO: 1: L36E, A40L, I41A, L42R, Q49M, Y52M, N65D,S68E, L70R, K73H, D77K, W79D, R81V, C87S, N96L, Y100D, L103Q, S105P,Y106E, S127V, K134W.
 16. The nucleic acid molecule of claim 13, whereinthe nucleic acid molecule encodes an hNGAL mutein comprises SEQ ID NO:53
 17. The nucleic acid molecule of claim 13, wherein the nucleic acidmolecule encodes an hNGAL mutein that has binding specificity forpyoverdine type III succinyl, pyoverdine type III succinamid, and/orpyoverdine type III α-ketoglutaryl.
 18. The nucleic acid molecule ofclaim 13, wherein the nucleic acid molecule encodes an hNGAL mutein thatis capable of binding pyoverdine type III complexed with iron with aK_(D) of about 200 nM or lower.
 19. The nucleic acid molecule of claim13, wherein the nucleic acid molecule encodes an hNGAL mutein comprises12 or more mutations at positions 36, 40, 41, 42, 49, 52, 65, 68, 70,73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQ IDNO:
 1. 20. A nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising a human neutrophilgelatinase-associated lipocalin (hNGAL) mutein polypeptide havingbinding specificity for pyoverdine type III, wherein the hNGAL muteincomprises 15 or more mutations at positions 36, 40, 41, 42, 49, 52, 65,68, 70, 73, 77, 79, 81, 87, 96, 100, 103, 105, 106, 127, and 134 of SEQID NO:
 1. 21. A host cell containing a nucleic acid molecule of claim20.
 22. The nucleic acid molecule of claim 20, wherein the nucleic acidmolecule encodes an hNGAL mutein comprises 15 or more of the followingmutations of SEQ ID NO: 1: L36E, A40L, I41A, L42R, Q49M, Y52M, N65D,S68E, L70R, K73H, D77K, W79D, R81V, C87S, N96L, Y100D, L103Q, S105P,Y106E, S127V, K134W.
 23. The nucleic acid molecule of claim 20, whereinthe nucleic acid molecule encodes an hNGAL mutein comprises SEQ ID NO:53.
 24. The nucleic acid molecule of claim 20, wherein the nucleic acidmolecule encodes an hNGAL mutein that has binding specificity forpyoverdine type III succinyl, pyoverdine type III succinamid, and/orpyoverdine type III α-ketoglutaryl.
 25. The nucleic acid molecule ofclaim 20, wherein the nucleic acid molecule encodes an hNGAL mutein thatis capable of binding pyoverdine type III complexed with iron with aK_(D) of about 200 nM or lower.
 26. A nucleic acid molecule comprising anucleotide sequence encoding a polypeptide comprising a human neutrophilgelatinase-associated lipocalin (hNGAL) mutein polypeptide havingbinding specificity for pyoverdine type III, wherein the hNGAL muteincomprises SEQ ID NO:
 53. 27. The nucleic acid molecule of claim 26,wherein the nucleic acid molecule encodes an hNGAL mutein consists ofSEQ ID NO:
 53. 28. A host cell containing a nucleic acid molecule ofclaim
 26. 29. The nucleic acid molecule of claim 26, wherein the nucleicacid molecule encodes an hNGAL mutein that has binding specificity forpyoverdine type III succinyl, pyoverdine type III succinamid, and/orpyoverdine type III α-ketoglutaryl.
 30. The nucleic acid molecule ofclaim 26, wherein the nucleic acid molecule encodes an hNGAL mutein thatis capable of binding pyoverdine type III complexed with iron with aK_(D) of about 200 nM or lower.